Virginia LIVINGSTON : Cancer Therapy -- Articles & US Patent 4692412 ( Method of preparing an autogenous vaccine )

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Virginia Livingston
Cancer Therapy

Claimed up to 83% Success Rate

A. Cantwell : Virginia Livingston, MD : Cancer Quack Or Medical Genius?
Wikipedia : Virginia Livingston
Unproven Methods of Cancer Management : Livingston-Wheeler Therapy
Livingston-Wheeler Therapy
V. Livingston : Toxic Fractions Obtained from Tumor Isolates...
V. Livingston : A specific type of organism cultivated from malignancy: bacteriology and proposed classification
V. Livingston : Some cultural, immunological, and biochemical properties of Progenitor cryptocides
Gumshoe News : Virginia Livingston’s Cancer Cure
M. Maxwell : Cancer Book
Livingston-Wheeler Therapy
E. Addeo : The Woman Who Cured Cancer
V. Livingston-Wheeler : The Conquest of Cancer
V. Livingston : US4692412 -- Method of preparing an autogenous vaccine
D. Mager Bacteria and cancer: cause, coincidence or cure? A review

http://www.rense.com/general72/cancer.htm

Virginia Livingston, MD : Cancer Quack Or Medical Genius?
By Alan Cantwell, MD

Cancer is the most frightening human disease and its cause remains elusive. Therefore, it seems inconceivable that the discovery of a germ cause of cancer would provoke such hostility among the cancer establishment. But, in truth, the belief in a cancer germ has always been the ultimate scientific heresy.

In the long history of cancer research there was never a physician more outspoken and controversial than Virginia Wuerthele-Caspe Livingston (1906-1990). For more than 40 years she championed the revolutionary idea that bacteria caused cancer and devised a treatment to try and combat these microbes by immunotherapy.

Sixteen years after her death she is now largely forgotten but still condemned by such powerful organizations as the American Cancer Society-and blacklisted on Quackwatch-a self-proclaimed "non-profit corporation dedicated to combating health-related frauds, myths, fads, and fallacies. "

LIVINGSTON'S CANCER RESEARCH

Beginning in the late 1940s, Livingston was able to grow bacteria from cancer tumors; and when she and her associates injected cancer bacteria into laboratory animals, some developed cancer. Other animals developed degenerative and proliferative diseases, and some animals remained healthy. Livingston believed the "immunity" of the host was an important factor in determining whether cancer would develop.

Virginia Livingston MD
(1906-1990)

In 1969 at a meeting at the New York Academy of Sciences , Livingston and her colleagues proposed that cancer was caused by a highly unusual bacterium which she named Progenitor cryptocides-Greek for 'ancestral hidden killer.' Neverthless, Livingston claimed elements of the microbe were present in every human cell. Due to its biochemical properties, she believed the organism was responsible for initiating life and for the healing of tissue-and for killing us with cancer and other infirmities. Critics of this research continued to insist there was no such thing as a cancer germ.

In her attempt to use a variety of modalities (diet, supplements, antibiotics, as well as traditional methods) to treat cancer, she utilized an 'autogenous' vaccine derived from the patient's own cancer bacteria found in the urine and blood. Livingston explained it was not an anti-cancer vaccine, but rather a vaccine to help stimulate and improve the patient's own immune system. The administration of this unapproved vaccine caused a furor in the cancer establishment and eventually legal action was undertaken against her and the Livingston-Wheeler Clinic in San Diego. In spite of all her legal troubles, she continued seeing patients until her death at 83.

In March 1990, the year of her death, a highly critical article on the Livingston-Wheeler therapy appeared in the American Cancer Society-sponsored CA: A Cancer Journal for Physicians. (No authors were listed.) The report advised patients to stay away from the San Diego clinic and claimed: "Livingston-Wheeler's cancer treatment is based on the belief that cancer is caused by a bacterium she has named Progenitor cryptocides. Careful research using modern techniques, however, has shown that there is no such organism and that Livingston-Wheeler has apparently mistaken several different types of bacteria, both rare and common, for a unique microbe. In spite of diligent research to isolate a cancer-causing microorganism, none has been found. Similarly, Livingston-Wheeler's autologous vaccine cannot be considered an effective treatment for cancer. While many oncologists have expressed the hope that someday a vaccine will be developed against cancer, the cause(s) of cancer must be determined before research can be directed toward developing a vaccine. The rationale for other facets of the Livingston-Wheeler cancer therapy is similarly faulty. No evidence supports her contention that cancer results from a defective immune system, that a whole-foods diet restores immune system deficiencies, that abscisic acid slows tumor growth, or that cancer is transmitted to humans by chickens." (The full report is on-line at:

http://caonline.amcancersoc.org/cgi/reprint/40/2/103)

BACTERIA AS A CAUSE OF CANCER

The recognition of disease-producing bacteria allowed medical science to emerge from the dark ages into the era of modern medicine. In the late nineteenth century when diseases like tuberculosis (TB) , syphilis, and leprosy were proven to be caused by bacteria, some doctors also suspected human cancer might have a similar cause.

The idea that bacteria cause cancer is considered preposterous by most physicians. However, despite the antagonistic view of the American Cancer Society and medical science, there is ample evidence in the published peer-reviewed literature that strongly suggests that 'cancer microbes' cause cancer.

Intracellular variably-sized coccoid forms in breast cancer.
Acid-fast stain; Magnification x1000, in oil

According to reports by Livingston and various other researchers, cancer is caused by pleomorphic, cell wall deficient bacteria. The various forms of the organism range in size from submicroscopic virus-like forms, up to the size of bacteria, yeasts, and fungi. In culture and in tissue the bacterial forms are variably 'acid-fast' (having a staining quality like TB bacteria). These bacteria are ubiquitous and exist in the blood and tissues of all human beings (yet another 'heresy'). In the absence of a protective immune response, these cell wall deficient bacteria may become pathogenic and foster the development of cancer , autoimmune disease, AIDS, and certain other chronic diseases of unknown etiology.

Needless to say, all this research fell on dead ears because bacteria were totally ruled out as the cause of all cancers in the early years of the twentieth century. Thus, bacteria observed in cancer were simply dismissed as elements of cellular degeneration, or as invaders of tissue weakened by cancer, or as 'contaminants' of laboratory origin.

LIVINGSTON AND PROGENITOR CRYPTOCIDES

Beginning in1950, in a series of papers and books, Livingston and her co-workers claimed the cancer microbe was a great imitator whose various pleomorphic forms resembled common staphylococci, diphtheroids, fungi, viruses, and host cell inclusions. Yet if the germ were studied carefully through all its transitional stages, it could be identified as a single agent. She was the first to suggest that the acid-fast stain was the key to the identification of the cancer microbe in tissue and in culture; and also demonstrated its appearance in the blood of cancer patients, by use of dark-field microscopy.

Anyone who takes the time to read Livingston's reports in the medical literature will quickly recognize that she was a credible research scientist, who allied herself with other experts-and was certainly not the quack doctor pictured by her detractors. Her achievements in cancer microbiology can also be found in her autobiographical books: Cancer, A New Breakthrough (1972); The Microbiology of Cancer (1977); and The Conquest of Cancer (1984). Her research has been confirmed by other scientists, such as microbiologist Eleanor Alexander-Jackson, cell cytologist Irene Diller, biochemist Florence Seibert, and dermatologist Alan Cantwell, among others.

Intracellular bacteria in prostate cancer.

Acid-fast stain; magnification x1000, in oil.

THE CANCER MICROBE AND BACTERIAL PLEOMORPHISM

Microbiologists have long resisted the idea of bacterial pleomorphism, and do not recognize or accept the various growth forms and the bacterial 'life cycle' proposed by various cancer microbe workers. Most bacteriologists do not accept the idea of a bacterium changing from a coccus to a rod, or to a fungus. Depending on the environment, the microbe in its cell wall-deficient phase may attain large size, even larger that a red blood cell. Other forms are submicroscopic and virus-sized. Electronic microscopic studies and photographs of filtered (bacteria-free) cultures of the cancer microbe show virus-size elements of the cancer microbe that can revert into bacterial-sized microbes.

The cancer microbe has adapted to life in man and animals by existing in a mycoplasma-like or cell wall deficient state. In tissue sections of cancer stained for bacteria with the special acid-fast stain, the microbe can be seen as a variably acid-fast (blue, red, or purple-stained) round coccus or as barely visible granules . At magnifications of one thousand times (in oil), these forms can be observed within and also outside of the cells.

Careful study and observation of the tiny round coccoid forms in cancer tissue indicate they can enlarge progressively up to the size of so-called Russell bodies, which are well-known to pathologists. Russell bodies can attain the size of red blood cells, and even larger.

William Russell was a well-respected Scottish pathologist who in 1890 first reported the finding of 'cancer parasites' in the tissue of all the cancers he studied. However, modern pathologists deny that Russell's bodies are microbial in origin. For more information on Russell bodies and Russell's 'cancer parasite' (and its intimate relationship to cancer microbes), Google: The forgotten clue to the bacterial cause of cancer; or go to:

http://www.joimr.org/phorum/read.php?f=2&i=50&t=50.

OVERLOOKING HIDDEN BACTERIA IN CANCER

Once bacteria were eliminated as a cause of cancer a century ago, it became dogma and impossible to change medical opinion. In this current era of medical science, one would think it impossible for infectious disease experts and pathologists to not recognize bacteria in cancer. However, bacteria can still pop up in diseases in which they were initially overlooked.

When a new and deadly lung disease broke out among legionnaires in Philadelphia in July 1976, two hundred twenty-two people became ill and thirty-four died. The cause of the killer lung disease remained a medical mystery for over five months until Joe McDade at the Leprosy Branch of the CDC detected unusual bacteria in guinea pigs experimentally infected with lung tissue from the dead legionnaires. Further modification of bacterial culture methods finally allowed the isolation of the causative and previously overlooked bacteria, now known as Legionella pneumophila.

Lymph node showing Hodgkin's lymphoma. Arrows point to variably-sized round coccoid forms and larger Russell bodies. Gram stain; magnification x1000, in oil.

Yet another example of dogma-defying research is provided by recent studies proving that bacteria (Helicobacter pylori) are a common cause of stomach ulcers, which can sometimes lead to stomach cancer and lymphoma. For a century, physicians refused to believe bacteria caused ulcers because they thought bacteria could not live in the acid environment of the stomach. In 2005 the Nobel Prize in Medicine was awarded to two Australian researchers for their 1982 discovery. These stomach bacteria could only be detected by use of special tissue stains. The CDC now claims that H. pylori causes more than 90% of duodenal ulcers and 80% of gastric ulcers. Approximately two-thirds of the world's population is infected with these microbes.

In the past four years there have been medical reports of newly discovered bacteria in serious lymph node disease; in Hodgkin's lymphoma; in cancer of the mouth; and in prostate cancer, to name only a few.

All these studies prove bacteria can pop up in diseases where they are least expected. Such a caveat is appropriate for doctors who think they know everything about cancer and who pooh-pooh all aspects of cancer microbe research.

A CENTURY OF CANCER MICROBE RESEARCH

Livingston never claimed that she was the discoverer of the microbe of cancer. In her writings she always gave credit to various scientists, some dating back to the nineteenth century, who attempted to prove that bacteria cause cancer. Some of these remarkable researchers include the long-forgotten cancer microbe studies of Scottish obstetrician James Young, Chicago physician John Nuzum, Montana surgeon James Scott, the infamous psychiatrist and cancer researcher Wilhelm Reich, microscopist Raymond Royal Rife, and others too numerous to mention.

This cancer microbe research has been explored in my books The Cancer Microbe: The Hidden Killer in Cancer, AIDS, and Other Immune Diseases [1990] and in Four Women Against Cancer: Bacteria, Cancer, and the Origin of Life [2005]-the story of Livingston, Alexander-Jackson, Diller and Seibert-four outstanding women scientists who attempted to bring the cancer microbe to the attention of a disinterested medical establishment. I was privileged to have met all these remarkable women, who greatly influenced my own cancer research.

Why is research exploring bacteria in cancer so strongly opposed? Perhaps it poses a threat to the money interests involved in the established and orthodox treatment for cancer. Various forms of cancer treatment include surgery, radiation and chemotherapy. These therapies might have to be reevaluated if it were proven that cancer was an infectious disease.

SUGGESTIONS FOR FURTHER INTERNET STUDY

Further information pertaining to cancer microbe research (both pro and con) can be found by Googling: cancer microbe; bacterial pleomorphism; cell wall deficient bacteria; "alan cantwell"; "virginia livingston"; "Eleanor Alexander-Jackson"; as well as other names and key words mentioned in this communication.

For a list of scientific publications pertaining to the microbiology of cancer, go to the PubMed website hosted by the National Institute of Health (www.ncbi.nlm.nih.gov) and type in "Cantwell AR", "Livingston VW", "Alexander-Jackson E", "Diller IC", "Seibert FB", etc. in the search box.

This short communication is unlikely to convince many health professionals that bacteria cause cancer. However, after four decades of studying cancer microbes in cancerous tissue, I am personally convinced that Dr. Virginia Livingston will one day be vindicated and recognized as one of the greatest medical geniuses of the twentieth century.

Ralph W Moss, cancer advocate and author of The Cancer Industry, notes her passing was "a major loss to the cancer world." In the Cancer Chronicles #6, 1990, he writes, "Virginia Livingston was a great person and a great scientist. Sadly, she never received the recognition she deserved in her lifetime. The true scope of her achievements will only become known in years to come."

This report honors the centennial of her birth which takes place on December 28, 2006.

BIBLIOGRAPHY:

Alexander-Jackson E. A specific type of microorganism isolated from animal and human cancer: bacteriology of the organism. Growth. 1954 Mar;18(1):37-51.

Cantwell AR. Variably acid-fast cell wall-deficient bacteria as a possible cause of dermatologic disease. In, Domingue GJ (Ed). Cell Wall Deficient Bacteria. Reading: Addison-Wesley Publishing Co; 1982. Pp. 321-360.

Cantwell A. The Cancer Microbe. Los Angeles: Aries Rising Press; 1990.

Cantwell A. Four Women Against Cancer. Los Angeles: Aries Rising Press; 2005.

Diller IC, Diller WF. Intracellular acid-fast organisms isolated from malignant tissues. Trans Amer Micr Soc. 1965; 84:138-148.

Greenberg DE, Ding L, Zelazny AM, Stock F, Wong A, Anderson VL, Miller G, Kleiner DE, Tenorio AR, Brinster L, Dorward DW, Murray PR, Holland SM. A novel bacterium associated with lymphadenitis in a patient with chronic granulomatous disease. PLoS Pathog. 2006 Apr;2(4):e28. Epub 2006 Apr 14.

Hooper SJ, Crean SJ, Lewis MA, Spratt DA, Wade WG, Wilson MJ. Viable bacteria present within oral squamous cell carcinoma tissue. J Clin Microbiol. 2006 May;44(5):1719-25.

Nuzum JW. The experimental production of metastasizing carcinoma of the breast of the dog and primary epithelioma in man by repeated inoculation of a micrococcus isolated from human breast cancer. Surg Gynecol Obstet. 1925; 11;343-352.

Russell W. An address on a characteristic organism of cancer. Br Med J. 1890; 2:1356-1360.

Russell W. The parasite of cancer. Lancet. 1899;1:1138-1141.

Sauter C, Kurrer MO. Intracellular bacteria in Hodgkin's disease and sclerosing mediastinal B-cell lymphoma: sign of a bacterial etiology? Swiss Med Wkly. 2002 Jun 15;132(23-24):312-5.
Scott MJ. The parasitic origin of carcinoma. Northwest Med. 1925;24:162-166.

Seibert FB, Feldmann FM, Davis RL, Richmond IS. Morphological, biological, and immunological studies on isolates from tumors and leukemic bloods. Ann N Y Acad Sci. 1970 Oct 30;174(2):690-728.

Shannon BA, Garrett KL, Cohen RJ. Links between Propionibacterium acnes and prostate cancer. Future Oncol. 2006 Apr;2(2):225-32. Review.

Wuerthele Caspe-Livingston V, Alexander-Jackson E, Anderson JA, et al. Cultural properties and pathogenicity of certain microorganisms obtained from various proliferative and neoplastic diseases. Amer J Med Sci. 1950; 220;628-646.

Wuerthele-Caspe Livingston V, Livingston AM. Demonstration of Progenitor cryptocides in the blood of patients with collagen and neoplastic diseases. Trans NY Acad Sci. 1972; 174 (2):636-654.

Young J. Description of an organism obtained from carcinomatous growths. Edinburgh Med J. 1921; 27:212-221.

https://en.wikipedia.org/wiki/Virginia_Livingston

Virginia Livingston

Born    1906
Died    1990
Citizenship    American
Nationality    American
Fields    Cancer

Virginia Livingston (1906–1990) was an American physician and cancer researcher who advocated the unsupported theory that a specific species of bacteria she named Progenitor cryptocides was the primary cause of cancer in humans. Her theories about P. cryptocides have not been duplicated by researchers, and a clinical trial of her therapy did not show any efficacy in the treatment of cancer. The American Cancer Society, which did not support Livingston’s treatment protocol for cancer, categorically denied her theory of cancer origins.

Life

Virginia Livingston was born Virginia Wuerthele in Meadville, Pennsylvania in 1906.

Both her father and grandfather were physicians and she also pursued a degree in medicine. Prior to attending medical school, Livingston earned three BA degrees in English, history, and economics from Vassar College. She then attended New York University, Bellevue Medical College and in 1936, received her degree in medicine. She was one of four women in her graduating class.[1]

Shortly after graduation, Livingston became the first female resident physician at a New York hospital where she was assigned to treat prostitutes infected with venereal diseases. While there, Livingston became interested in the study of tuberculosis and leprosy, and later scleroderma, a disease affecting the tissues and skin. After studying scleroderma tissues with the darkfield microscope, she claimed to find an acid-fast organism that consistently appeared in her slides. Thinking that scleroderma had some characteristics that were like cancer, Livingston then began studying malignant tissues and subsequently claimed to find evidence of acid-fast organisms in every sample. It was this early research that prompted the young physician to devote her career to the study of a specific microorganism involved in cancer.

Early research

In 1946, Livingston published a paper in which she stated she had established that a bacterium was a causative agent in scleroderma.[2] In 1947, she cultured a mycobacteria-like organism in human cancer and, according to her peer-reviewed paper, fulfilled Koch's postulates establishing an apparent cause and effect.[3] In 1949, Livingston was named chief of the Rutgers-Presbyterian Hospital Laboratory for Proliferative Diseases in New Jersey where she continued her cancer research.[4] It was during this time that Livingston formed a lifetime association with Dr. Eleanor Alexander-Jackson of Cornell University. Jackson's specialty was the study of mycobacteria and particularly, the species responsible for tuberculosis. Jackson had developed specific culture media for growing the microbe and a technique for observing it known as the "triple stain" because she felt this microbe wasn't amenable to conventional modes of culturing and microscopy.

Livingston and Jackson also collaborated on work on the Rous sarcoma virus (RSV) at Lederle Laboratories. Livingston claimed that when RSV cultures were passed through special filters designed to hold back all but the smallest virus particles, she was able to grow bacteria; this was considered a controversial claim since bacteria are considerably larger than viruses and are not supposed to exist in filtered RSV serum. After healthy animals were exposed to the Rous bacterial filtrates, Livingston and Jackson claimed that cancerous lesions developed.[5] This finding led to speculation that such bacteria could be transmitted from poultry to humans and this became a primary reason Livingston ordered her cancer patients to not eat poultry while they underwent her treatment. Scientists have since rejected Livingston findings, arguing there is no evidence supporting her claim.[6]

In 1956, Livingston published a paper suggesting a causative bacterium in Wilson's disease.[7] In 1965, she reported isolation of a variably acid-fast mycobacterium in patients with myocardial vascular disease. During this time, she also began a small test trial of anti-bacterial vaccines made from the body fluids of cancer patients and reported moderate success.[8] Between the years 1965-1968, Livingston received Fleet Foundation and Kerr Grants, and continued her investigation into a bacterial cause of human cancer. She also published a paper describing the presence of a substance identified as Actinomycin-D which she said could damage chromosomes and promote cancer.[9]

In 1969, Livingston and her husband Afton Munk Livingston, established the Livingston-Wheeler Clinic in San Diego, California, and began formally treating cancer patients. The therapeutic program included autogenous vaccine made from killed bacteria derived from body fluids; a low sodium diet consisting of organic foods, fruits and vegetables high in a substance Livingston called "abscisic acid"; immune enhancing vaccines (gamma globulin, BCG) and antibiotics. Livingston prescribed antibiotics after cross testing them with patients' cultures to see which had the most antibacterial activity. Livingston also recommended that patients not consume poultry products based on her earlier research.

After her husband’s death, she married Owen Webster Wheeler, one of the first patients she claims to have successfully treated for head and neck cancer. Shortly after, the clinic was renamed the Livingston-Wheeler clinic. In 1970, Livingston officially named her cancer organism Progenitor cryptocides, and presented her findings to the New York Academy of Sciences.[10] According to her biography, Progenitor was a pseudonym meaning "ancestral" and the name was chosen because Livingston believed the microbe existed as early as the Precambrian era, and it was an endegenous component of life itself. The name "cryptocides" was a Greek and Latin word which meant "hidden killer". The microbe was classified under the order Actinomycetales. Livingston described Progenitor as an intermittently acid-fast mycobacterium that displayed highly variable growth cycles. According to Livingston the microbe was pleomorphic, and had cell wall-deficient and filter-passing forms resembling viruses, with the ability to adopt a variety of shapes including spindles, rods and cocci.[10]

1974-1990

In 1974, Livingston published a paper which described her isolation of human chorionic gonadotropin (hCG) from cancer bacteria.[11] She then advanced one of her central hypotheses.

Livingston theorized that hCG is both a component of human cancer, but also innately involved in embryonic growth and fetal survival.[4] She wrote that hCG is saturated in the placenta, and blocks the mothers’ antibodies from attacking the fetus, partly made of foreign DNA (and not recognized by host immunity). By the same token, hCG performs a similar function in cancer, conferring protection to malignant tissues. Livingston believed that after Progenitor hybridizes with cancer cells, it imparts an ability for them to produce hCG in a manner similar to that of the developing fetus. Based on this duality of function, Livingston called hCG “the hormone of life and the hormone of death”. She also stipulated that vaccines which target hCG-producing bacteria could also halt the progression of cancer. And she claimed that absicins could also neutralize hCG.

Controversy

Though some bacteria have been associated with cancer[12] (for instance H. pylori has been associated with stomach cancer[13]) Livingston's postulated relationship between cancer and P. cryptocides was never proven in several follow up studies conducted by independent investigators. Researchers confirmed that bacteria provided by Livingston produced hCG, but several other studies demonstrated that numerous bacteria in both cancer patients and healthy individuals also produced the substance.

Occurring before the existence of techniques to analyze DNA, Livingston and other investigators' ability to differentiate bacteria based on morphology and chemical characteristics was limited. However, even given technological limitations at the time, Livingston's classification methods were described as full of "remarkable errors", attributing characteristics to Actinomycetales (the order Livingston believed P. cryptocides belonged to) shared by no other members of the order. Some evidence supports P. cryptocides is the result of a mistaken identification of a Staphylococcus strain of bacteria and later studies of the samples provided by Livingston proved to be Staphylococcus epidermidis and Streptococcus faecalis.[6]

The American Cancer Society (ACS) did not support Livingston’s treatment protocol for cancer, and has categorically denied her theory of the cancer bacterium P. cryptocides the primary cause of human cancer. The ACS also challenged the efficacy of Livingston’s autogenous vaccine and concluded in its report that there was no corroboration of either P. cryptocides or the efficacy of her autologous vaccine.[6] Since Livingston hadn’t stocked earlier cultures of her alleged microbe, it is not possible to decipher precisely what those cultures contained.

Clinical testing

A case-control study using self-selected, matched but not randomized groups with late stage cancer compared survival and quality of life between cancer patients receiving conventional treatment and those undergoing the Livingston-Wheeler therapy. The results were reported in The New England Journal of Medicine in 1991, and found no differences in survival among patients whether treated conventionally, or via Livingston's treatment. The NEJM report also stated that when comparing the two groups, the "quality of life were consistently better among conventionally treated patients from enrollment on". Based on this trial, the ACS deemed Livingston's cancer therapy without efficacy, and considered it an "unproven therapy".[14]

While both groups of patients in the trial deteriorated at equal rates---all in effect dying of their disease---patients in the Livingston-treated group were reported to have had a "poorer quality of life" at the start of the trial.[14] The study's lead investigator, Barrie Cassileth, acknowledged that "the University of Pennsylvania patients had a significantly better quality of life at all times, including enrollment" and that, quality of life "was different at base line", with Livingston's patients rated worse.[14] Patients in both treatment arms also received conventional therapies in addition to Livingston's therapy. Livingston's patients also received BCG during the trial---an FDA-approved cancer adjuvant which has been found effective for several cancers, including those of the bladder and colon.

At the study's conclusion, Barrie Cassileth commented:

"This study...involved only patients with diagnoses and stages of disease for which there is no effective conventional treatment. Therefore, the results cannot be generalized to patients with less advanced stages of disease or to other treatment regimens." Cassileth also said, her study group "hypothesized that survival time would not differ between the two groups on the basis of the assumption that the unproved remedy would be no more effective with end-stage disease than conventional care, itself largely ineffective".

Death

Shortly after speaking before an Office of Technology Assessment (OTA) hearing on alternative cancer therapies and attending her 60th reunion at Vassar College in 1990, Livingston accompanied her daughter Julie Anne Wagner on a European trip. She developed chest pains while visiting the Greek islands and then succumbed to heart failure in Athens on June 30, before being transported to a Paris Hospital.[15]

References

Lerner, Michael G. (1994). "Chapter Sixteen: Virginia C. Livingston--Integrating Diet, Nutritional Supplements, and Immunotherapy". Choices in healing: integrating the best of conventional and complementary approaches to cancer. Cambridge, Mass: MIT Press. ISBN 0-262-62104-5.

Wuerthele-Caspe, V; Brodkin, E; Mermod, C (1947). "Etiology of scleroderma; a preliminary clinical report". The Journal of the Medical Society of New Jersey. 44 (7): 256–9. PMID 20248313.

Livingston, V (1947). "Microorganisms associated with Neoplasms". New York Microscopial Society Bulletin. 2 (2).

Addeo, Edmond G.; Virginia Livingston-Wheeler (1984). The conquest of cancer: vaccines and diet. New York: F. Watts. ISBN 0-531-09806-0.

Wuerthele-Caspe, V (1955). "Neoplastic infections of man and animals". Journal of the American Medical Women's Association. 10 (8): 261–6. PMID 13242416.

"Unproven methods of cancer management: Livingston-Wheeler therapy". CA: A Cancer Journal for Clinicians. American Cancer Society. 40 (2): 103–108. 1990. doi:10.3322/canjclin.40.2.103. PMID 2106368.

Wuerthele-Caspe, V; Alexander-Jackson, E; Gregory, M; Smith, LW; Diller, IC; Mankowski, Z (1956).
"Intracellular acid-fast microorganism; isolated from two cases of hepatolenticular degeneration". Journal of the American Medical Women's Association. 11 (4): 120–9. PMID 13306623.

Livingston, V (1965). "Mycobacterial Forms in Myocardial Vascular Disease". The Journal of the Am Med Women's Association. 20 (5): 449–452.

Wolter, G.; Livingston, A.; Livingston, V.; Alexander-Jackson, E. (1970). "Toxic fractions obtained from tumor isolates and related clinical implications". Annals of the New York Academy of Sciences. 174 (2): 675–689. Bibcode:1970NYASA.174..675L. doi:10.1111/j.1749-6632.1970.tb45590.x. PMID 5278141.

Livingston, VW; Alexander-Jackson, E (1970). "A specific type of organism cultivated from malignancy: bacteriology and proposed classification". Annals of the New York Academy of Sciences. 174 (2): 636–54. Bibcode:1970NYASA.174..636L. doi:10.1111/j.1749-6632.1970.tb45588.x. PMID 5278140.

Livingston, VW; Livingston, AM (1974). "Some cultural, immunological, and biochemical properties of Progenitor cryptocides". Transactions of the New York Academy of Sciences. 36 (6): 569–82. doi:10.1111/j.2164-0947.1974.tb01602.x. PMID 4530542.

Mager, D. L. (2006). "Bacteria and cancer: cause, coincidence or cure? A review". Journal of translational medicine. 4: 14. doi:10.1186/1479-5876-4-14. PMC 1479838 Freely accessible. PMID 16566840.

Peter, S.; Beglinger, C. (2007). "Helicobacter pylori and gastric cancer: the causal relationship". Digestion. 75 (1): 25–35. doi:10.1159/000101564. PMID 17429205.

Cassileth, B. R.; Lusk, E. J.; Guerry, D.; Blake, A. D.; Walsh, W. P.; Kascius, L.; Schultz, D. J. (1991). "Survival and Quality of Life among Patients Receiving Unproven as Compared with Conventional Cancer Therapy". New England Journal of Medicine. 324 (17): 1180–1185. doi:10.1056/NEJM199104253241706. PMID 2011162.

Moss, R (1990). "The Cancer Chronicles". 6.

http://onlinelibrary.wiley.com/doi/10.3322/canjclin.40.2.103/abstract
DOI: 10.3322/canjclin.40.2.103
CA: A Cancer Journal for Clinicians; Volume 40, Issue 2, March/April 1990, Pages 103–108

Unproven Methods of Cancer Management : Livingston-Wheeler Therapy

https://www.ncbi.nlm.nih.gov/pubmed/2106368
CA Cancer J Clin. 1990 Mar-Apr;40(2):103-8.

Livingston-Wheeler therapy

Abstract

Livingston-Wheeler's cancer treatment is based on the belief that cancer is caused by a bacterium she has named Progenitor cryptocides. Careful research using modern techniques, however, has shown that there is no such organism and that Livingston-Wheeler has apparently mistaken several different types of bacteria, both rare and common, for a unique microbe. In spite of diligent research to isolate a cancer-causing microorganism, none has been found. Similarly, Livingston-Wheeler's autologous vaccine cannot be considered an effective treatment for cancer. While many oncologists have expressed the hope that someday a vaccine will be developed against cancer, the cause(s) of cancer must be determined before research can be directed toward developing a vaccine. The rationale for other facets of the Livingston-Wheeler cancer therapy is similarly faulty. No evidence supports her contention that cancer results from a defective immune system, that a whole-foods diet restores immune system deficiencies, that abscisic acid slows tumor growth, or that cancer is transmitted to humans by chickens.

http://onlinelibrary.wiley.com/doi/10.1111/j.1749-6632.1970.tb45590.x/abstract
Ann. N.Y. Acad. Sci., Volume 174, Unusual Isolates from Clinical Material, Pages 675–689 ( Oct 1970 )

TOXIC FRACTIONS OBTAINED FROM TUMOR ISOLATES AND RELATED CLINICAL IMPLICATIONS*

http://onlinelibrary.wiley.com/doi/10.1111/j.1749-6632.1970.tb45588.x/abstract
Annals of the New York Academy of Sciences. 174 (2): 636–54.
doi:10.1111/j.1749-6632.1970.tb45588.x.

A specific type of organism cultivated from malignancy: bacteriology and proposed classification

Livingston, VW; Alexander-Jackson

http://onlinelibrary.wiley.com/doi/10.1111/j.2164-0947.1974.tb01602.x/abstract
Transactions of the New York Academy of Sciences. 36 (6): 569–82.
doi:10.1111/j.2164-0947.1974.tb01602.

Some cultural, immunological, and biochemical properties of Progenitor cryptocides

Livingston, VW; Livingston, AM

Abstract

Guinea pigs became skin-positive to test doses of PPD (purified protein derivative-Seibert) and to other related PPD preparations in various degrees following immunization with phenolized cultures of Progenitor cryptocides, an intermittently acid-fast pleomorphic filterable Actinomycete isolated from human cancer patients. This reactivity, indicative of the relationship to M. tuberculosis as well as to several other related microbes, may account for the effective treatment of some types of human cancer with BCG (Bacillus Calmette Guérin). Another property of P. cryptocides is the production in vitro of a parahormone immunologically and biologically related to human chorionic gonadotropin. Since the cancer patient often exhibits various types of hormonal imbalance, the microbic exogenous production of this hormone may explain the neoplastic parahormone syndrome in man. There are some biological differences in experimental animals between the microbic and human chorionic gonadotropin, but the in vitro and radioimmunological tests are identical. The microbic hormone appears to play an important role in human cancer, since it not only is present in tumors, body tissues, and fluids but may be excreted in large amounts in the urine. The amount excreted is variable, depending upon the rate of microbic production of the hormone in vivo and the resistance of the host as expressed through immunological and metabolic degradation systems.

https://gumshoenews.com/2017/05/16/virginia-livingstons-cancer-cure/

Virginia Livingston’s Cancer Cure

During the last hundred years, a surprisingly large number of doctors have found ways to cure or alleviate cancer. These ways are not the Big Three – surgery, radiation, and chemo – that are the ones almost exclusively recommended by the profession.

Let’s begin with Virginia Livingston (1906-1990), a graduate of Bellevue Medical School, daughter of Herman Wuerthele, MD (1885-1967). In her first book, Cancer: A New Breakthrough (1972), she claimed a success rate of 82%. Here are four cases:

D.K. – Age 71, operated on for carcinoma of prostate, followed by removal of testicle, 1966. He had multiple spinal metastases and arthritis of many joints. He was barely able to move around. He was placed on autogenous vaccine and mandelamine, 1 gram four times a day with dietary and vitamin adjuvants. Previous to his prostatic surgery he had a bowel resection for cancer of the colon. At the present time the spinal metastases have healed, he says he has no evidence of arthritis, is in perfect health and works.

J.M. — Age thirty-five, had a left radical mastectomy March 3, 1965, when four months pregnant. Pathological diagnosis was infiltrating arcinoma, scirrhus and medullary types. After delivery of a normal child she had a hysterectomy May 28, 1965, and was placed on estrogen therapy from August 24, 1966, through January 9, 1967. Autogenous vaccine was made which she took for a year and intermittently since. This type of tumor is universally fatal. Her physician says she is in good health at the present time (1972) with no signs of recurrence.

F.B. — Male age twenty-seven from Utah, who was operated on for severe headaches after a number of convulsive seizures. The pathological diagnosis was astrocytoma, grade III to IV, infiltrating the surrounding area. He received anti-convulsants, radiotherapy and antibiotics. In 1966 when he was doing very badly and appeared to be terminal, he was placed on autogenous vaccines and mandelamine, one gram four times daily, plus vitamins and dietary supplements. He remained on this regimen for two years. The vaccine was discontinued in October 1970. His physician said there is no evidence of any tumor.

A Longshoreman, age 46, operated 1967, for a mass on the right side of his neck. Pathological diagnosis was malignant lymphoma, reticulum-cell type with invasion of all glands. These were not resectable because they extended under the sternocleidomastoid muscle. He received X-ray, 4500 R, in eighteen treatments. Since then he has had no other treatment except autogenous vaccine continuously with erythromycin 250 mgm twice a day. He says he is completely well and works full time on the docks.

Leprosy the Clue

After World War II, Virginia worked in a New York hospital and saw many cases of TB (tuberculosis) and leprosy. Note: every physician’s experience is unique. It is incorrect to think that all doctors possess the same knowledge; much depends on who happens to walk into their office one fine day.

One fine day into Virginia’s office (she was a school doctor) walked the school nurse, complaining of ulcers on the fingers, a perforation in the septum (the piece of cartilage that separates the two nostrils), and hardening of the skin. This was in 1947. Her own doctor had given her a diagnosis of scleroderma.

Virginia associated the symptoms with leprosy as the patient reported that she could not feel hot or cold on the affected skin. Virginia Livingston decided to do some lab work on this case. She took smears from the woman’s nose and the ulcers on her hands and stained them with the stain used for identifying both leprosy and TB, namely a “Ziehl-Neelsen” stain.

Peering into the microscope, Virginia saw the same type of microorganisms one sees in leprosy. She treated this patient with the medication used for lepers, and the skin healed. Later, Virginia gave the same medication to other scleroderma patients and it worked!

Whatever she saw in the microscope that day became central to her later theory that cancer is explainable by bacteria. That has not been widely accepted. But she made a separate discovery that did later become standard in science. Namely, she found that bacteria can and do secrete a hormone, human chorionic gonadatropin, hCG, which is essential for human life. Hooray!

In a Nutshell, Livingston’s Theory

Virginia believed that cancer is not a foreign visitor. It is part of our body from birth and it is never going to go away. Cancer is characterized by mitosis, the dividing and replication of cells. Cell division itself is not to be despised; it is the basis of our initial growth in childhood, and occurs as part of the repair work that steadily goes on in the body. When a piece of skin gets scraped off, you just wait for it to regrow. We need cell division!

If cell division gets out of control, however, it may make tumors. Tumors are bunches of new cells that don’t associate in the normal manner with surrounding cells and have no purpose. A cancer doctor has the title “oncologist” from the Greek word onco for mound. Virginia never became a “moundologist.”

She surmised that a tumor happened because the person’s immune system was not functioning as it normally does. As for the cancer microbe that she believed to be ever-present in our body, she gave it the name Progenitor cryptocides (crypto=stealth; cide=to kill).

The Livingston Program for Treating Cancer

Virginia Livingston does not claim to have invented the bacterial theory of cancer. Others such as William Russell and Royal Rife, she notes, got there first.

Now have a look at what she prescribes: she tells the cancer patient to get his Progenitor cryptocides back under control. That is something that, in a healthy person, is taken care of by the immune system.

When your immune system sees the cryptocides microbes going where they shouldn’t go, she says, it treats them as invaders and acts to protect you. The immune system is ever-alert for the non-normal, and can do what must be done.

Run-of-the-mill miracles.

One cancer patient, a physician named Owen Wheeler, was cured by Virginia, and subsequently married her. They established the Livingston-Wheeler Clinic in San Diego, and helped thousands of persons.

But what if your immune system is not working well and can’t call up the right response? Then a tumor may form. Stuff may also travel around your body and metastasize.

What should the doctor do? She will try to get your immune system working again.

Livingston’s treatment program has two prongs:

Use nutrition to build up the immune system. She advises fresh fruits, vegetables, and nuts (nothing out of a can). No meat or dairy until you are recovered. Lots of Vitamins A and C, and

Vaccinate the patient with the antigen he needs. Material for that vaccination comes from the patient himself; his urine is used to culture the bacteria which are then made into an autogenous vaccine. In some cases she also gives antibiotics. She often gives a blood transfusion, from a family member.

“Getting” Virginia Livingston

Virginia was still working at her clinic at age 83 when the government closed it down. The feds and most states do that to any doctor who dares defy the rule to use only the Big Three cancer cures. (surgery, chemo, radiation).

A few months after that, she expired.

Dr Livingston constantly made her patient’s progress available for inspection by the medical authorities. She also arranged for a random survey of the records, going so far as to hire an outsider to choose 62 cases under a meticulous set of guidelines. Yet when she published the survey no one was wiling to read it.

In the back of her book you will find a section with the pitiful heading “Ten Cases That I Wish Someone Would Investigate.”

In 2001, Saul Green wrote a bad evaluation of Livingston for Sloan Kettering, making nary a mention of cures she wrought for 20 years! He did, however, importantly remind us that it is a felony in California to treat a cancer patient with unapproved methods.

Cantwell and Pleomorphism

One physician who came to Livingston’s aid was Los Angeles dermatologist Alan Cantwell, MD.

He had already published his clinical finding of a cancer microbe, in 1968, before he got to know Livingston. Cantwell spends much time at an ordinary microscope using an “oil immersion lens” that allows him to see what most pathologists claim they don’t see. He credits a Spanish microbiologist for giving him the clue to cancer’s similarity to tuberculosis:

“[Conrado] Xalabarder totally transformed my concept about how tuberculosis-causing mycobacteria reproduce and grow and drastically change their appearance.”

Ah, changing appearance – and behavior – and size, and – who knows? Maybe their species identity – is the name of the game for the bacteria we are concerned with here.

The pioneering taxonomist, Carolus Lineaus, born 1707 in Sweden, grouped animals together in phyla based on shared characteristics, for example, he put Homo sapiens into the phylum chordata, as we share the characteristic of having a backbone. (Sigh. If only we had backbone, and not just a backbone!)

When a bacterium is not encased in the normal way by a cell wall (that is, when it’s CWD – cell-wall deficient), it has potential to sneak around and get up to no good. Harken to this:

“There is increasing evidence that CWDB and CWD fungi are often associated with endocarditis, septicemia, meningitis, pneumonia, and infections of bones and joints. When prompt diagnosis is critical, it is helpful to include examination for CWD microorganisms as part of the first laboratory study.”

That’s from an article that Lida Mattman, and her co-author Mehnga S. Judge, contributed to Domingue’s Cell Wall-Deficient Bacteria. (1982: 440). Amazingly, it did not spark eager research as to its rather glaring potential for meningitis cases.

PATENT

Patent #4, 692, 412 (expiry date 2006) was awarded to V. Livingston, and Afton Livingston, and Eleanor Alexander-Jackson for the making of an autogenous vaccine from the blood or urine of cancer patients. Their application for the patent claimed:

“All cancerous bloods examined have revealed the cryptocides organisms. [It] is apparently ubiquitous in nature, existing in a reservoir in soil and water, and is found in all classes of animals…. It can exist as a latent infection in host tissue without causing apparent ill effects. However, when the immunologic barriers are lowered it can invade the host in prodigious numbers and involve any or all of the host tissues, causing various kinds and degrees of pathologic change.” [Note: Award of patent does not mean claim proven.]

This Patent’s Method for Making Autogenous Vaccine:

“Obtain a midstream clean-catch specimen of urine in a sterilized screw-top glass container. Make up DiFco’s brain-heart infusion agar: 37 grams of the agar base are added to a liter of distilled water heated to melt and mix, and distributed into flasks or bottles of 95 ml amounts, and autoclaved. Five percent (5%) human blood … is added when the melted agar has cooled down to 45-50 degrees C., and the mixture is poured into sterile Petri dishes. Streak the surface of the blood agar plate with a sterile swab dipped in the urine. Incubate plate to 37 degrees C. and examine after 24 hours.

“If growth has appeared, note types of colonies, make duplicate smears, and stain one by Gram’s stain and the other by Alexander-Jackson’s modified Ziehl-Neelsen technique: flood slide with Kinyoun’s carbolfuchsin for 3 to 5 minutes in the cold, wash, decolorize briefly with 70% alcohol containing 1 to 3% HCl as these organisms decolorize more readily than M. tuberculosis, counter stain by flooding slide with Loeffler’s methylene blue and add 6 to 8 drops of normal (4%) sodium hydroxide. Tilt slide to mix, and wash after 30 seconds.”...

https://www.mskcc.org/cancer-care/integrative-medicine/herbs/livingston-wheeler-therapy

Livingston-Wheeler Therapy

Clinical Summary

Livingston-Wheeler Therapy is an alternative approach to cancer treatment that gained popularity from the 1970s to early 1990. It used several treatments to supposedly stimulate the immune system, including a strict vegetarian diet, BCG vaccine, coffee enemas, autogenous vaccine, vitamins, antibiotics, antioxidants, nutritional counseling, and support groups/counseling. The regimen was based on the theory that cancer was caused by the bacterium Progenitor cryptocides, which developer Virginia Livingston-Wheeler reported to have isolated in a wide variety of cancer tissues (3) (4). A weakened immune system would then allow the bacterium to grow, and consequently the therapy’s focus was immune-stimulation. Although a number of viruses and bacteria have been associated with various cancers (8), a link between the bacteria named by Livingston-Wheeler and cancer was never confirmed by independent researchers.

A self-selected, matched-cohort, prospective comparison of patients at Livingston-Wheeler Clinic and a conventional cancer center found no difference in survival times between groups, but did find consistently lower quality of life in the Livingston-Wheeler cohort (6).

Metabolic diets may result in nutrient deficiencies (5). Repeated use of coffee enemas has been linked to several deaths from serious infection and electrolyte imbalance (2). In a 1990 position paper, the American Cancer Society urged cancer patients not to use these treatments (1).

Mechanism of Action

Although a number of viruses, bacteria, and parasites have been associated with various cancers (8), a link between the bacteria named by Livingston-Wheeler and cancer was never confirmed by independent researchers. Rather, independent analyses of cultures provided by Livingston-Wheeler identified these bacteria as Staph epidermis, Strep faecalis, Staph faecalis, and other unrelated bacteria (1).

In a 1990 position paper, the American Cancer Society urged cancer patients not to use this treatment, as no evidence supports its efficacy (1). In addition, although the Livingston-Wheeler diet was similar to recommendations made by the American Cancer Society, its nutrient deficits, especially for calcium, iron, vitamins D and B12, and protein, would be unsuitable for some cancer patients.

Adverse Reactions

Common (metabolic diet): Nutrient deficiencies (calcium, vit B12, protein), anemia, and malabsorption may result from metabolic diets (1).

Reported (autogenous vaccine): Malaise, aching, slight fever, and tenderness at injection site (1).

Case Report (Coffee enemas): Death attributable to fluid and electrolyte imbalance causing pleural and pericardial effusions after use of coffee enemas, 4 per day for 8 weeks (2).

https://www.amazon.com/Woman-Cured-Cancer-Livingston-Wheeler-Cancer-Causing/dp/1591203724

The Woman Who Cured Cancer: The Story of Cancer Pioneer Virginia Livingston-Wheeler, M.D., and the Discovery of the Cancer-Causing Microbe

by
Edmond G Addeo

This story is now more relevant than ever as the latest science is now validating the protocols of Dr. Livingston-Wheeler who will one day be placed in the same class as Pasteur, Curie, Salk/Sabin and their discoveries.

https://www.amazon.com/conquest-cancer-Dr-Virginia-Livingston-Wheeler/dp/1482097109

The Conquest of Cancer

by Dr. Virginia Livingston-Wheeler (Author), Edmond G. Addeo (Contributor)

This is the original book published in 1985 on the pioneering work of Dr. Virginia Livingston-Wheeler. It is being republished to make it widely available to the general public.

Quotes from the original book: "Dr. Virginia Livngston-Wheeler is one of the great, unsung scientists of present day medicine. When her discovery of the 'cancer microbe' becomes... accepted, she will undoubtedly be known as the Pastuer of this century." Alan R. Cantwell, Jr. M.D.

"Dr. Virginia has long been pioneering in new concepts of cancer cause and treatment... the many successess she has had in treating advanced cancer patients demonstrate what can be accomplished when a physician in cooperation with understanding patients tries to expand the limits of cancer treatment." Ray G. Crispen, Ph.D

"This important book could very well save your life or the life of someone you love. Dr. Virginia Livingston-Wheeler has succeeded in devising a scientifically rational program for the prevention and treatment of cancer." Richard A. Kunin, M.D.

US4692412
Method of preparing an autogenous vaccine

Inventor(s):    LIVINGSTON VIRGINIA, et al.

A method of preparing an autogenous vaccine for use in improving the immunocompetence of animals affected with neoplastic disease characterized by the production of choronic gonadotropin by the microorganism Progenitor cryptocides, ATCC 31, 874.

DESCRIPTION OF THE INVENTION

This invention relates to chemical substances comprising a product of the growth of a microorganism of Order II, Actinomycetales Buchanan, as identified in Bergey's Manual of Determinative Bacteriology, Sixth Edition, 1948, p. 111, to method of preparing same and isolation thereof and to the chemotherapeutic treatment of animals and humans therewith. Published works have identified additional species and resulted in a classification of the organism under the order Actinomycetales. This order has been reclassified as follows:
ORDER--ACTINOMYCETALES
family--Progenitoraceae
genus--Cryptocides
species--
Cryptocides tumefaciens
Cryptocides sclerodermatis (sclerobacillus)
Cryptocides wilsonii
stains isolated from lupus erytheuratosis, rheumatoid, arthritis, periarthritis, nodosum, sarcoidoses (that is, from collogen diseases other than cancer and other such diseases specified elsewhere herein)
varieties: hominis, rodentii, avii, etc. A culture of Progenitor cryptocides was deposited in the American Type Culture Collection, Accession Number 31,874.

All of the species noted above have been observed to be interchangeable within the scope of this invention.

P. Cryptocides has been assayed and assigned the tentative formula, C30 H38 N2 O3 (certain products of Cryptocides were crystals extracted from cultures of urine from terminal cancer patients). Extracts also have been crystallized from the blood and urine of cancer patients and the crystals result from the presence of the organism.

The microorganism involved (Cryptocides) has been identified as a highly pleomorphic intermittently acid-fact micro-organism, with both a virus-like and a PPLO or L transitional phase. This organism is a great simulator, whose various forms may resemble micrococci, diphtheroids, bacilli, fungi, viruses, and host-cell inclusions. Cryptocides has the ability to change its form and may vary its appearance from that of a fungus to that of a cluster of virus-size pleuro-pneumonialike organisms (PPLO or Mycoplasma). Collagenophillic mycobacterium-like, which include the cancer organism, are able to change their forms. Cryptocides has filterable or extremely small forms (submicroscopic) similar to viruses, and rather large mycelia. There were some variations as to size and some differences in the kind of media or material in which it will grow. Certain strains of it ferment one kind of sugar, some others, and some can live with little (or no) oxygen, whereas some require more. It can be identified as a single agent. The microorganism undergoes many changes in morphology and some of those forms might be zoogleal or "L" Forms. Zoogleals are intermediate forms of microorganisms which ordinarily have cell walls, but, in which, under certain circumstances the cell walls are absent. Some of these forms can be passed through very fine filters that hold back the usual bacteria and allow only very small particles such as viruses and small L forms to pass through. Such filter-passing bodies can regrow to become bacilli (bacterial cultures). The microoganisms involved have many forms but they always grow up to be the same thing no matter how often they are cultured. Cryptocides is not a virus but is a pleomorphic bacterium.

Cryptocides is acid fast, that is, it retains the Ziel-Neelsen stain in the presence of acid. Cryptocides is related to the tuberculosis family of microbes. It is filterable through filters designed to hold back bacteria. It is sensitive to tetracycline, kanamycin, ampicillin and furacin, but occasionally resistant to pencillin, sulfa drugs and mycostatin. As for the pleomorphism, cryptocides exists as virus-sized bodies of 20 to 70 microns, as elementary bodies of 0.2 micron, and in coccoidal forms of 0.5 microns or larger. The latter are usually gram-positive and resemble common micrococci but are distinguishable by variation in size and the sprouting of filaments or spicules. The organism may also appear in amorphous mycoplasmalike forms, as rods or filaments of varying lengths, and in older cultures, as spores and hyphae.

Cultures made from animal tumors and fluid have great similarities with cultures derived from many types of fresh uncontaminated human tumors, from blood and other body fluids of patients who have advanced cancer.

The L-forms are bacterial forms without cell walls. They resemble pleuro-pneumonilike organisms (PPLO), also known as mycoplasma. However, the mycoplasma appear to reproduce continously under some conditions in the same stage, with the absence of cell walls, while other organisms have a tendency to revert more quickly to the more stable bacillary or coccal forms of origin. The L-forms are the link between bacteria and the viruslike minute bodies that are a stage in the life cycle of certain microorganism. Many viruses may actually be L-forms of microbes which, under certain conditions may be induced to return to their original forms. Previously the appearance of the both adult and L-forms led to the erroneous conclusion that there was a mixture of microorganisms, a contamination of pure strains with other nonrelevant microorganisms, but this was shown to be an erroneous misconception. Some true contaminants are readily recognized by their growth pattern but the Cryptocides is a great simulator of other organisms. It requires infinite patience to observe its growth pattern and to recognize its transition from one form into another.

The microorganism involved requires definitive bacteriologic media for its primary isolation, differential staining techniques for its identification, high power microscopic resolution, and the electron microscope to reveal its most minute forms. Specific cultures can be obtained on solid media used for the isolation of the tubercle bacillus. This mycobacterium-like organism is believed to be a primary etiologic agent in proliferative and degenerative diseases such as cancer and perhaps many other socalled autoimmune diseases. Cryptocides is believed to be the causative or infectious agent (microbial) of cancer, in all of its forms, Cryptocides would therefore be called an antigen. More specifically, the filterable forms of P. Cryptocides which are of virus size are the causative agents in human and animal cancers.

Peyton Rous did not call his tumor filtrates (from chickens) viruses but instead "tumor agents." His material could be dried and held on a shelf at room temperature for months and then, mixed with saline, it could be reactivated to initiate fresh tumors. A true virus has been defined as a submicroscopic infectious unit that lives only in the presence of living cells and cannot exist even momentarily outside of them. Many have tried to find a virus implicated in some form of human cancer, but none has been found.

Applicants have grown the Rous tumor agent in sterilized beef broth that contained suitable nutrients for bacterial growth, and traced its growth pattern through all of the bacteriological stages. Applicants knew that the infectious agent passed through filters that permitted only the passage of so-called viruses. Applicants filtered the cultures, not the extracts of the tumors, through bacteria restraining filters and studied these with a electron microscope. Applicants kept the cultures in which there did not appear to be any visible form of life, incubated them at 37 DEG C. and from these seemingly clear broths with the agent in them, there arose the bacterial and fungal stages of the cryptocides. Applicants ruled out contamination that might account for the bacterial growth on incubation by repeating the experiment dozens of times. It was a tedious process but proved that this so-called virus could and did convert to a bacterium that had not only submicroscopic forms but also bacillary, coccal or round forms, and that could also develop funguslike stages and spores. (On studying the growth of the tubercle and lepra bacilli these stages were entirely comparable with the Cryptocides.)

When applicants examined the cultures obtained from human cancers, there was no discernible difference in the growth pattern. The growth pattern of the chicken cancer isolates and that of man were the same. They grew in the same kind of broth in the same way and they appeared the same in chicken and human tissues. They had the same staining properties with the Ziehl-Neelsen dye. Applicants did sheep immunization studies in which they found significant cross-agglutination between the Rous sarcoma, fowl leukosis and various strains of human cancers. When applicants injected the isolated cultures into mice, the characteristic disease and lesions developed. The Rous isolates had to be readapted to chicken tissues by passage on the allantoic membrane of fertile eggs and then replanted into young chicks. Applicants also carried on immunization of rabbits with the leuokosis agent and used the antiserum to cure chickens dying of fowl leukosis. In every way the Rous agent appears to be a prototype for human cancer.

The above studies led to the cultivation of the same kind of microorganisms from other animal tumors. These invariably grew and appeared similar to the Rous and human strains. Sometimes there were differences in size or different sugar or oxygen requirements for cultivation, but essentially they were the same basic type or organism.

The cancer organisms (cryptocides) appear to resemble mycoplasma, organisms that exist without cell walls, expecially since the cytosinguanine ratio of their mucleic acid, DNA, is similar to that of certain mycoplasma. However, the usual mycoplasma tend to remain in their state of existence without cell wals but the Cryptocides may pass rapidly through the stage without walls to the form of true bacteria. Perhaps all mycoplasma could be induced to become bacteria but this is still a disputed point.

Dr. Robert Huebner, head of the National Institute of Allergy and Infectious Diseases, Bethesda, Md., has stated that cancer is a viral infection. Of the various agents suspected, he stated that the C-particle is the most likely agent. It has been called by this name because the round bodies found in cancerous tissues often appear in a C shape. However, the comparison of the C-particles in mouse leukemia with the filted Cryptocides isolates examined under the electron microscope show them to be similar in size and shape. In preparations from cultures the round forms are often seen to split and assume the C form. It would seem that this splitting into a C is characteristic, but not necessarily a method of identification. All the other methods are necessary as well.

The cancerous growth itself is not the entire disease. The small coccuslike granules which can be seen dividing in cancer cells represent the intracellular parasite that is the causative agent. The parasite within the cancer transforms the normal cell into a sick cell that cannot mature by differentiation. It is the filtered material from tumors and other cancerous growths, as well as the cells themselves, which transmit cancer from one species to another species. Or, in other words, the cancerous agent can cross species barriers and infect other species.

P. Cryptocides not only causes cancer but a number of other ailments that effect man. The infectious nature of arthritis, of some kinds of heart, liver and kidney impairment, and most recently of diabetes is known. The patterns of these diseases point to their latent infectious nature. Cryptocides is an infectious agent. But, the tumors are only a part of the resultant disease. In addition to tumors, there are cheesy lesions or areas resembling tuberculosis, which can involve any one of the essential organs such as the liver, kidney heart or lung. These organs might show changes in the connective tissue, called collagen, which can lead to degeneration as seen in the chronic human degenerative diseases. The organism may assume a latent form and be inactive as long as the body's defense mechanisms are adequate, but when they are not, disease results. The exact kind of disease depends on the age of the host and its state of resistance, as well as the strain of the organism.

All cancerous bloods examined have revealed the cryptocides organisms. Applicants have made a film of untreated blood from a terminal cancer patient in which the parasites are seen in Brownian motion in the red cells. The parasites stay inside the cells of patients who are holding their own against the disease, but in advanced cases, the numerous cells rupture, releasing the organisms.

The microorganism is apparently ubiquitous in nature, existing in a reservoir in soil and water, and is found in all classes of animals, including man. It can exist as a latent infection in host tissue without causing apparent ill effects. However, when the immunologic barriers are lowered it can invade the host in prodigious numbers and involve any or all of the host tissues, causing various kinds and degrees of pathologic change equilibrated between the ferocity and numbers of the invader and the ability of the host to resist them. Not only is the organism pleomorphic but the pathologic changes induced in experimental animals show varying degrees of disease ranging from the lethal through the semi-immune, neoplastic and degenerative stages. Hyper-immune and degenerative stages may be relatively quiescent but can become slowly and progressively fatal.

Man and/or animal can be a latent carrier of the Cryptocides. Many of applicants' experimental animals that have surived cancer have developed interstitial collagen disease as a result of their inoculations with applicants' bacterial isolates of Cryptocides, and also developed heart lesions. When baby mice born of infected mothers died, the autopsy showed destruction of heart muscle. These lesions contained the acid-fast organisms (Cryptocides) in the heart muscle. Also, a number of research people in England have reported strange microbic bodies previously unrecognized in the hearts of people who died of coronary disease. One of the applicants had cancer of the forehead treated successfully with radium fifteen years previously, but was a latent carrier of the Cryptocides. That applicant was treated after that period of time with an autogenous vaccine, has had a new vaccine prepared every year, and has continued treatment.

"Mycobacterial Forms in Myocardial Vascular Disease", Virginia Wuerthele-Caspe Livingston and Eleanor Alexander-Jackson, (1965) proposes the theory that there are microbic bodies in the lesions of heart diseases and that they are especially numerous in the areas where the blood vessels have ruptured. Until recently the theory has been the coronary blood vessels of the heart are narrowed due to arteriosclerosis, and that fatty deposit in the wall of the vessels, and overweight are the determining factors in this type of heart disease. Now the medical researchers are becoming aware of the fact that the blood bessels themselves are often not involved so much as the supporting tissues and muscles of the heart so that the heart vessels rupture due to extrinsic factors outside the vessel rather than from intrinsic disease. This is particularily true of patients with collagen diseases such as scleroderma and lupus erythematosis. Vascular and myocardial pathology is related to chronic low-grade infection by the mycobacterium-like organisms (Cryptocides).

Degenerative changes occur in coronary heart disease in the presence of the invasive mycobacterial parasite cryptocides.

Postmorten heart sections of 6 patients with coronary and aortic disease were stained by the Fite modification of the Ziehl-Neelsen technique (for demonstrating Lepra bacilli in sections) using Kinyoun's carbon-fuchsin, and compared with sections of the same involved areas stained with conventional H and E. Eight predominant types of lesions were observed in the myocardium

1. PERIVASCULAR CHANGES AROUND THE SMALL CORONARY VESSELS. In the loose connective tissues numerous small acid-fast bodies can be seen.

2. CELLULAR INFILTRATION. This is frequently seen not only around the vessels but between the muscle fibers as well. These cells consists almost entirely of mononuclear types, predominantly lymphocytes, while large mononuclear phagocytes laden with organisms plasma and other mononuclear cells are present in relatively large number.

3. FIBROBLASTIC INFILTRATION. The presence of these organisms appears to stimulate the formation of fibroblasts. In some areas, the muscle fibers and interstitial tissues appear to be replaced by fibroblasts.

4. INFARCTION. Where there has been an infarct, there may be a softened central area with numerous small acid-fast cocci and coccobacilli present in the collagenous hemorrhagic softened area. 5. NECROSIS. Necrotic changes may involve the blood vessels. Striking degenerative changes of the vessel walls are observed as illustrated not only by the sections of coronary vessels but also by the sections of in involved aorta. Proliferative changes may involve the endothelium, with invasion of the endothelial cells, and are accompanied by thickening and narrowing of the wall. Hairlike filaments of the organisms were seen protruding into the lumen. These changes are also present in the vasa vasorum of the aorta.

6. THROMBOSIS AND RECANALIZATION. Some areas of recanalization were observed in heart, liver, and spleen.

7. CHANGES IN THE ELASTIC LAYER OF THE AORTA. The elastic fibrils have lost their identity and have become collagenized with loss of structure. As scar tissue forms, cholesterol-like plaques occur. It seems possible that deposity may be derived in part from the fatty envelopes of these organisms. In other tissue where masses of the organisms have proliferated, polyhedral crystals resembling cholesterol have been observed.

8. CHANGES IN THE HEART MUSCLE. Individual nuclei of the heart muscle are frequently parasitized, and replaced by small acid-fast globoidal bodies. The muscle fibers themselves appear in a state of gradual digestion and disintegration by both minute and larger acid-fast forms.

All of the above can be treated and detected by this invention

Neoplastic changes have been shown by Diller and Diller (Intracellular acid-fast organisms isolated from malignant tissues, Trans. Amer. Micr. Soc., 84:138-148, 1965), to arise in tissue culture as the result of exposure to this specific invading microorganism.

The Journal of the American Medical Association, July 28, 1969 Vol. 209, No. 4, contains a summary of the work of K. A. Bisset, New Scientist, June 12, 1969, who speculates that many diseases like leukemia and arthritis could be caused by Mycoplasma or by forms of this elusive bacteria and wrote that the fact that mycoplasm can break down into viruslike particles, easily identifiable on electron-microscope examination and similar to those found in blood of leukemia patients, leads to a strong suspicion that Mycoplasma may be a culprit in the development of certain malignant processes.

Dr. Florence B. Seibert, Veterans Administration Research Laboratory at Bay Pines, Fla., has reported immunologic studies with the organisms. Labeled antiglobulin, which was specific for an isolate from a human breast, adenocarcinoma induced specific fluorescence in the white blood cells of patients with leukemia and myeloma, demonstrating an immunologic relationship.

Koch's law is the foolproof method of proving the cause of a disease. It is as follows:
1. The microorganism must be present in every case of the disease.
2. It must be possible to cultivate the microorganism outside the host in some artifical media.
3. The inoculation of this culture must produce the disease in a susceptible animal.
4. The microorganism must then be reobtained from these inoculated animals and cultured again.

Applicants have fulfilled Koch's law using pure, uncontaminated cultures of cryptocides. Pure cultures were obtained repeatedly from the various proliferative and neoplastic diseases of both men and animals. Then they were injected into animals capable of being infected. Gradually diseased areas developed which resembled those from which the cultures were obtained. Then the pure cultures were reisolated from the infected animals. Thus Koch's postulates were fulfilled.

A blood specimen of a terminal cancer patient was cultured, the culture was extracted and the extract produced tumors in mice. This demonstrates the growth factors.

In one attempt to produce antibodies and antiserum in sheep that would be beneficial in the treatment of human cancer, sheep were immunized with an attenuated or weakened culture. Twenty sheep were examined and found to be free of disease. Some of the stock cultures applicants had on hand such as cultures from human breast cancer, from a sarcoma of a young boy, from a human leukemia, from the Rous chicken tumor, from arthritis, and from fowl leukosis, were attenuated. Applicants injected two sheep with each strain. After about four weeks, some of the sheep became sick. Attenuated vaccines from the cancer cultures were used weekly for immunization. Several ewes aborted their young. The fetuses were macerated. Some of the sheep developed very swollen painful joints and could scarcely graze. Others looked poorly because emaciated. Applicants realized that the vaccines which were attenuated were still alive and had not fully immunized the sheep but had diseased them. The sheep were bled in order to assay their serum for antibodies. Sera was obtained but the sheep had to be destroyed. Although the sheep had to be destroyed applicants learned that the fowl leukosis serum agglutinated in high dilution the cultures from the boy's sarcoma, that the breast cancer serum reacted with the human leukemia isolates, and that the Rous sarcoma serum reacted with all of the cultures. This meant that the cultures from the human cross-reacted with one another strongly and with the animal sera, showing that tumors are not tissue or species specific.

Three chickens having fowl leukosos, a cancerous disease, and which could no longer stand up, were taken to applicants' laboratory and in a short time they were dead. Applicants made cultures from their heart's blood. These grew to be the same kind of cultures as those derived from all of the other tumors experimented with by the applicants.

A pure, selectively grown bacterial culture of the type described above, obtained from urine and blood using sterile precautions, contains chemical substances related and/or identical to the actinomycin group. To test this point, a phenolized pure culture was acidified with HCl to pH 2 (Congo red/thymol blue) and was left standing overnight after short boiling. A mixture of n-butanol/conc. NaCl, equal parts by volume, containing a few drops of glacial acetic acid, was used for extraction. After gentle shaking for about 15 minutes a dark cherry-red layer of the solvent was separated for further processing. This crude mixture gave peak absorption at 440/450 mn and 410/425 mn values which compare favorably except for a third absorption peak at 240 m.mu. obtained by Waksman (Waksman, S. A., and Lechevalier, H. A.: The Actinomycetes, vol. III, The Williams & Wilkins Co., Baltimore, 1962, p. 168) which was missing. From an ascending paper chromatogram a reddish zone was eluted with ethylacetate and an acetone-ether mixture, both gave upon evaporation some microscopic crystals (red plates). The controls containing broth and 2% phenol but no organism gave upon extraction a barely yellow-tinted layer of the solvent. This exploratory separation technique was then repeated with several 25-hour urine specimens obtained from terminal cancer patients. After separation of the organic layer from the urine specimens again a more or less pronounced color was present ranging from dark honey-brown to cherry red; controls taken from healthy persons did not contain such colors. The presence of these dark colored compounds seems to be most pronounced in terminal cases. All crude mixtures isolated from cultures and/or urine were subjected to further separation by chromatographic techniques.

METHODS FOR THE ISOLATION AND IDENTIFICATION OF A PLEOMORPHIC INTERMITTENTLY ACID-FAST ORGANISM FROM NEOPLASTIC DISEASES, AND THE PREPARATION OF CULTURES

Isolation from Urine (Crofton Method)

Obtain a midstream clean-catch specimen of urine in a sterlized screw-top glass container.

Make up DiFco's brain-heart infusion agar: 37 grams of the agar base are added to a liter of distilled water, heated to melt and mix, and distributed into flasks or bottles of 95 ml amounts, and autoclaved. Five percent (5%) human blood (outdated bloodbank blood may be used) is added when the melted agar has cooled down to 45-50 degrees C., and the mixture is poured into sterile Petri dishes.

Streak the surface of the blood agar plate with a sterile swab which has been dipped in the urine. Incubate plate to 37 degrees C. and examine after 24 hours. If growth has appeared, note types of colonies, make duplicate smears, and stain one by Gram's stain and the other by Alexander-Jackson's modified Ziehl-Neelsen technique: flood side with Kinyoun's carbolfuchsin for 3 to 5 minutes in the cold, wash, decolorize briefly with 70% alcohol containing 1 to 3% HCl as these organisms decolorize more readily that M. tuberculosis, counter stain by flooding slide with Loeffler's methylene blue and add 6 to 8 drops of normal (4%) sodium hydroxide. Tilt slide to mix, and wash after 30 seconds.

APPEARANCE OF THE MICROORGANISMS ON BLOOD AGAR

Colonies--usual types of growth obtained

1. white discoidal, often hemolytic, and with a raised center--having a fried egg appearance, but usually larger than classic PPLO colonies grown on PPLO agar.

2. grayish muccoid, often confluent.

3. pigmented: yellowish, occasionally pinkish coral.

4. wrinkled intermediate SR worm-casting type resembling M. tuberculosis colony.

5. dull granular surfaced, irregular edges, often hemolytic, and resembling B. subtilis, but virulent for mice. Motile forms transferred tp A-J broth produce a white or grayish white soft rim or pellicle, and a toxin-like substance.

This organism tends to resist emulsification to some degree when a loop of culture is rubbed with a drop of water on a glass slide to make a smear.

MICROSCOPIC APPEARANCE

The cancer isolate is either Gram positive or Gram variable. The Gram stain is not the stain of choice, but should be used to eliminate true Gram negative organisms, which show no Gram positivity at all such as B. coli, proteus or pseudomonas.

Acid-fast forms may or may not be found, as this organism is intermittently acid-fast rather than more consistently so as in M. tuberculosis or other classic mycobacteria. However, if possible, careful search for acid-fast forms is desirable, as they are hallmarks of this mycobacterium-like organism. Slender filaments, sometimes with lateral branching, and sometimes acid-fast, help to distinguish it from common micrococci. The rods may be slender and diphtheroidal, or thicker and subtillis-like. The latter sometimes contain tiny brightly acid-fast bodies surrounded by a colorless vacuole-like area or capsule. This appearance plus virulence for mice and guinea-pigs distinguish them from subtillis rods. The main morphologic forms are:

1. tiny acid-fast elementary-type bodies, often refractile;

2. Coccoidal forms of varying sizes with or without threads or protruding filaments;

3. rods as described above; X, Y, and V forms commonly seen, filaments may become very long and wide;

4. cyst-like bodies of various sizes from 3 to more than 10.mu. and often containing smaller bodies which may be brightly acid-fast;

5. L or PPLO forms consisting of a lightly stained matrix containing more deeply stained bodies of various sizes, or tangled branching threads and ring-forms. These are revealed by Alexander-Jackson's Triple Stain modification of the Ziehl-Neelsen technique;

6. spore forms of oval shape seen in old cultures;

7. sub-microscopic bodies 20-70 m.mu. revealed by the electron microscope, and of virus size.

The product of the growth of the specific Actinomycetales organism is a chemical substance which is obtainable on recrystallization (Wolter)--of a suspension of the isolate with conventional paper chromatography annular separation procedures, and identifiable as comprising a formation of

(a) red crystals identical with those described by Waksman as having the absorption peaks indicated, supra;

(b) yellow crystals, similar to an actinomycin D fraction;

(c) formation of small placques of crystals similar in appearance to gramicidin;

(d) a waxy, higher-alcohol formation, and

(e) a brownish, foul aromatic residue.

CHROMATOGRAPHIC IDENTIFICATION

To an acidfied (pH 5) sample of urine in which the organisms are grown, (phase I) and kept in the refrigerator, is added one-fourth volume of n-butyl alcohol and the mixture is shaken for one-half hour. The mixture is refrigerated until the layers separate. Separation is done by decantation first and then by using a separatory funnel. The butanol layer has attained a reddish brown color and in some cases a yellowish color. This procedure results in an aqueous phase (II) and a butanol phase (III). The aqueous phase is extracted once or twice more in the same manner with n-butanol, so long as the color appears in the extract.

A portion of the combined extracts is evaporated in an evaporator at 35 DEG-40 DEG C. The dried residue is dissolved in a small amount of methyl alcohol, solution (IV). Layers of a silica gel preparation, MM-SGel-HR for thin layer chromatography, Machorey Nagel and Co. 516 Durem, Germany, is spread on glass plated, prepared, dried and stored in a dessicator. By means of a small pipette, a spot of solution (IV) is placed on the silica gel layer of one of these plates near one edge and near this spot at the same distance from this edge and in the same way, another spot of actinomycin D, (Merck, Sharp and Dohme), is placed in the same manner. Both spots appeared bright yellow. Other pairs of spots of these two solutions are placed on the same row using 5 m.mu., 10 m.mu. and 15 m.mu.. (One m.mu. equals one microliter.) Of the solvents tried for developing the chromatograph, the most effective was butanol-methanol-water in the ratios of volume of 6:1:3. 60 ml. of butanol, 10 ml methanol and 30 ml of distilled water are mixed and put into a thin layer chromatography chamber. The paper lining the walls of the chamber is wet by swirling the solution in the chamber. Then the plate with the spots is placed on edge in the chamber with the row of spots parallel to and near the bottom but above the surface of the solution. A cover is placed over the top of the chamber sealing it. The chamber is kept in the dark during the process of separation since a better yield elution is obtained. In previous runs each actinomycin D spot travelled as a single spot leaving nothing in the pathway by visible or ultraviolet light, and phase (III) left material spread from the top streaking down along the pathways. However the top was always at a level with and the same color as the actinomycin D. Subsequently, after drying the plates, the actinomycin D spots and those portions from the spots of phase (III) on a level with the actinomycin D spots are cut out and separately eluted with methanol, as are other portions from the spots of phase (III), since these may contain other actinomycins than actinomycin D. Visible ultraviolet and infrared absorption spectrograms are made of the different elutes and compared. The TLC method is based and adapted from methods reported by Cassani et al, J. Chrom. v. 13, 1964, 238-239.

ACTINOMYCIN BIO-ASSAY

Blood and tissue cultures and urine samples obtained in accordance with the foregoing procedures were extracted with and equal volume of butanol, water, acetic acid (4:5:1) and the upper phase taken to dryness. The residues were taken up into two 5 ml. portions of ether and evaporated at 35 DEG-40 DEG C. Paper discs were dipped into ether solutions of extract, the ether evaporated and the discs placed on standard actinomycin assay plates. A standard preparation of actinomycin D was also run. The zones of inhibition are shown in Table I; zones above 15 mm. in diameter fall within the standard range and are calculated in terms of actinomycin D equivalent.

TABLE I

Zone
Diameter (mins).
Act. D (ug/ml)
Broth Extracts
(1) 14 <0.2
(2) 15 0.2
(3) 16 0.4
(4) 12 <0.2
(5) 16 <0.4
(6) 14.5 <0.2
(7) NA <0.2
(8) NA <0.2
(9) 14 <0.2
(10) NA <0.2
(11) NA <0.2
(12) NA <0.2
(13) NA <0.2
(14) NA <0.2
(16) NA <0.2
Urine Extracts
(15) NA <0.2
(17) 22 4.2
(18) 16 0.35
(19) NA <0.2
(20) 18 0.8
(21) 17 0.5
(22) 13 <0.2
(23) NA <0.2

UV ABSORPTION CURVES

0.2 ml. of each ether solution was evaporated to dryness and the residue taken up in 1 ml. of methanol and UV absorption curves were recorded. End absorption at this level of purity prevented measurement of 440 mm. absorption. Small peaks, typical of trace amounts of actinomycin, were found with samples 1-6 inclusive and sample 8 with a slight response present with sample 17. Definitive biochemical tests such as the cytosine-guanine percentage of the DNA have helped to classify the cryptocides microbial isolates.

Further concerning cryptocides, these organisms, which appear primarily as small acid-fast granules in young cultures, and which tend to become non-acid-fast in the larger forms present in older cultures, may exhibit a number of morphologic phases with intermediate transitional forms. These include (1) filterable and submicroscopic bodies; (2) larger granules readily visible under the light microscope an often resembling ordinary micrococci; (3) larger globidal cystlike bodies and thin-walled sacs containing the smaller forms; (4) PPLO or L type zoogleal symplasms without cell walls; (5) rods of various sizes capable of developing a characteristic motility; (6) long filaments and threads which may show lateral branching; and (7) thick-walled spore-like bodies. The lesions produced by these organisms in experimental animals were generally pseudocaseous, degenerative in type, occasionally neoplastic, and occurred principally in the liver, kidneys, and lungs although at times, there was involvement of the heart, spleen adrenal glands, stomach, lymph nodes, and omentum.

Dr. Afton Munk Livingston, and Dr. Virginia Livingston, Transactions of the New York Academy of Sciences, May 1972, report the recognition of the P. cryptocides organisms in the blood of cancer patients compared with the blood of healthy individuals, of which a summary follows. Examinations by darkfield microscope of fresh blood, and also by brightfield microscope using supravital stains serving as a diagnostic and prognostic tool in following the course of the cancerous disease in the patient in conjunction with several other microbiological evaluations.

PREPARATION OF SLIDES FOR BLOOD EXAMINATION

The patient's finger is immersed in 70 percent alcohol and air dried. A steril lancet is used to puncture the finger, a small drop of free-flowing blood is placed on a sterile clean slide and covered with a sterile covership. Care is taken that the blood does not flow beyond the edge of the coverslip. Using a small weight for approximately one minute, light pressure is applied to the coverslip to spread and separate the blood cells. The preparation is then examined under darkfield at .times.750 and .times.1350 magnification. For lightfield examination, the same method is followed and in addition, a small drop of 1 percent aqueous sterile crystal violet, freshly prepared and filtered, gently applied to the preparation. If the number of organisms, to the blood as well as the motility of the various stages are to be evaluted, then the blood is diluted 1:100 with sterile distilled water using a sterile red-blood-cell diluting pipette. The pipette is then thoroughly shaken and a few drops are expelled from the pipette into a sterile Petri dish. A small measured amount of 1 percent aqueous crystal violet is added. This mixture may then be used to flood a blood counting chamber. This method provides a quantitative estimate of the numbers of the organism as well as their motility, which may last as long as fifteen minutes. However, for the usual brightfield examination of the blood with crystal violet, the blood drop is placed directly by the slide and the small amounts of crystal violet is added before the coverslip is placed over the preparation and light pressure applied.

DARKFIELD EXAMINATION OF UNSTAINED FRESH BLOOD PREPARATION

A number of interesting observations may now be made by the darkfield, pulsating orange bodies in the red cells may be observed. In the background, there are bright dancing forms which appear to be small L-forms of the organism. In several infected hosts a number of motile rods may be observed. Spheroplasts and mesosomes both large and small are present. These may have many fine delicate vibrating forms in their periphery. Forms resembling a medusa or a octopus with waving filaments may be present. Organisms may bud from the surface of the red cells and from fine hairlike filaments which resemble the handle of a tennis racquet. There may also be numerous threadlike filaments free in the serum, varying in size, some 10-15 microns in length. These are motile and appear to wind in and out around the red cells. There are also long tubular structures 50 microns or more in length, and about 10 microns in width that are milky white, highly luminescent, containing numerous refractile granules. The tube in some cases appears to arise from a coalescence of the L-forms or to bud from a spheroplast. It is transparent since cells can be seen through it. When the tube wall disintegrates the refractile bodies are released in the serum and may enter fresh red cells. There are also large round milky white forms appearing to be protoplasts about 20 to 60 microns in diameter which contain granules resembling spheroplasts or mesosomes. The protoplasts may have budding forms at the periphery and may release rather large vesicular refractile bodies resembling the spheroplasts or mesosomes. At times, the extruded mesosomes are large enough to be mistaken for red blood cells, but they do not have the bluish tinge of red cells seen in darkfield. Rather, minute dancing particles may later appear within them.

In addition, shrunked red cells with a ground-glass appearance spiculated at the periphery may be observed. We have termed these structures "spent cells" since they appear to be red cells that have been consumed by the parasites. They are lighter and smaller than normal erythocytes and have a tendency to be pushed to the periphery of the blood drop when it is prepared for examination. Changes in the character of the leukocytes are also apparent. Many leukocytes in the advanced stages of diseases appear smudged, inactive and only dimly luminescent whereas normal leukocytes have vigorously active granules and active amoeboid movements. Under some circumstances great numbers of fine spicules occur in the dark field. These are very delicate and appear to arise from minute L-forms. They are not thrombocytes. At times they appear to shed from the surface of the protoplasts. Why they should be more numerous at one time than at another is not understood but their appearance may be related to the pH of the blood. Orange crystalline forms of the organisms as well as free crystals may also be seen in and around the microbial clusters in the plasma. They apparently arise from the waxy-secretions of these mycobacteriumlike organisms. These are the crystals that have been extracted from pure cultures and urines of terminal cancer patients and that have been used for various types of bioassay.

BRIGHTFIELD EXAMINATION OF SUPRAVITALLY STAINED FRESH BLOOD PREPARATIONS

On a blood preparation stained with crystal violet and examined by the brightfield method a clear white light and a magnification of at least .times.1000 microbial forms are revealed that are not seen in the darkfield. There are large branching fungal forms that are not luminescent in the darkfield. These fungal forms may extend over a considerable area involving several microscopic fields. Some of these are branching and appear to have conidial or frutting bodies attached to their branches. Microcolonies may be clearly seen surrounding individual red cells and some appear to arise from parasites extruded from the cells. These microcolonies appear to develop into a network of interlacing branching fungal filaments which act as bridges between the red cells and cause them to adhere in clumps. The number of fungal forms which hold the erythrocytes together or adhere to their surfaces may be directly related to the sedimentation rate. The greater the adherence of the erythrocytes due to the mycelial forms, the more rapid the sedimentation rate. The red cells become separate and free as the number of both intra-and extracellular parasites diminish. The stained preparations in the counting chamber have L-forms, which appear much more numerous than in the darkfield, and occur in clusters, which have marked Brownian movement. These clusters agglutinate and become motionless after ten to fifteen minutes. Introduction of gamma globulin or specific antiserum under the coverslip of the counting chamber caused instant agglutination and cessation of motion. By this method, antibody activity of blood serum can be roughly estimated. Other dyed microbial forms in the brightfield may be compared with those in the darkfield. The vibrating orange bodies in erythrocytes in the darkfield appear as violet bodies in lightfield. The brightly luminous tubles take on a light violet color with deep purple granules.

The same comparison between darkfield and stained brightfield preparations may be drawn by examining blood cultures grown in broth. Hanging drops of cultures sealed with sterile vaseline are preferable to ordinary wet perparations since they are safer to handle and can be preserved for a longer period of time. Conventional staining of slide preparations appears to break up many of the delicate microcolonies and interlacing fungal forms. Wet supravitally stained preparations in hanging drops also indicate the degree of motility of many of the microorganisms. Other dyes have been used which penetrate to some extent but do not provide sufficient contrast. They are Sudan black, saffron yellow, Congo red, May Grunwald, toluidine blue, gentian violet, as well as several others.

All cancerous patients yielded L-forms as well as other pleomorphic stageson blood culture which, on further cultivation, developed the typical acid-fastness of the Progenitor cryptocides group as previously described. However, the cancer patient even in the advanced stages of the disease is usually afebrile. Comparable numbers of microorganisms other than the Progenitor cryptocides groups might be expected to produce an acute febrile reaction. There undoubtedly can be a mild or transitory bacteremia in blood due to relatively nonpathogenic bacteria such as some of the diphtheroids. However, with the previously described methods, the great numbers of the Progenitor group as a silent but lethal bloodstream infection may be readily demonstrated. Advancing infection of the bloodstream with P. cryptocides is relatively asymptomatic until large numbers of the organisms are present and there is a concomitant breakdown of the immunological and dextoxifying system.

The autogeneous vaccine is known to exist by disc-saturated inhibition on culture plates of extracts and also from the serum of cancer patients.

Administration of the autogenous vaccine should be initiated by high dilutions of the lowest order of dosage at twice weekly intervals, with gradually increasing dosages until overdosage symptoms occur. Preferably, subcutaneous injection of the autogenous vaccine in a suitable pharmaceutical carrier, such as sterile water or saline solution may be employed, although oral administration of the product in a suitable carrier also may be employed.

Use of the autogenous vaccine of this invention may prove to be of value in the palliative treatment of animals and humans afflicted with various forms of neoplastic diseases, as indicated by treatment thereof with autogeneous vaccines made from a suspension of the isolates in 2% phenol. In preparation, the vaccine is allowed to stand overnight at room temperatures, centrifuged and further diluted with 0.5 phenol or saline.

Subcutaneous injection is initiated with the highest dilution of 1 million organisms per ml and 0.1 ml twice weekly, until overdosage symptoms occur. Therapy is continued with higher concentrations, e.g., 10 million and 100 million organisms/ml. Oral administration of the same dosage can also be employed.

A study of one hundred random blood samples, taken in the office of a physician who specialized in allergy and immunologic disease, showed that all tumor-bearing patients, in comparison to office personnel used as controls, gave positive cultures for the cryptocides organism. A number of patients with chronic degenerative disease were also positive. While many patients who had reached a healthy old age were negative, several "tired" young people without apparent disease were positive.

A reddish brown material has been extracted from the tissue, urine and blood of cancer patients in increasing amounts as they became terminally ill, and (this material has not been found in normal controls. It is carcinogenic for mice, increasing the incidence of pulmonary tumors. The biological effects have been assayed in preliminary studies with tissue-culture systems and with tumor-genesis in mice.

The applicants have found the presence of actinomycin-like crystals in body tissues and in cultures.

The basic requirement for formation of the cancer cell is the causative microorganism; all other factors such as coal-tar irritants, other microorganisms, the aging process, any chronic irritants leading to poor local resistance and giving rise to immature, succeptible reparative cells, may prepare the living matter, e.g., for the multiplication of the cancer organism and its penetration into the cyptoplasm and nucleus of the host cell.

Apparently the organism cryptocides can invade both cytoplasm and nucleus of host cells in any type of host tissue when body defenses are lowered. In experimental animals it can cause lesions that appear as necrotic abscesses, granulomas, fluid-filled cysts of neoplasms. The type of lesion apparently depends on specific and nonspecific immunocompetence and the age of the host.

Certain chemicals can have a tremendous effect upon the entire hormonal system. One of these substances is actinomycin produced by several of the actinomyces organisms and probably by many of the Actinomycetales. There is a whole array of chemicals and biologic produced by this group of microbes, which have been used as antibiotics and antineoplastic agents in some cases. The actinomycins even in very high dilution of one part in a billion or more may have a profound effect upon the entire business of life. The important thing to remember is that no funtcion of the body is exempt from this toxic material which is produced by these microorganisms belonging to the Actinomycetales. Not only are the normal functions of the host's hormonal system deranged but there are "false or counterfeit hormones" produced with further throw the body off balance. There is a practice of castrating men and women to arrest the growth of cancer. If castration is successful in prolonging life, then adrenalectomy and pituary gland removal might be done when the effects of tumor inhibition from the castration have worn off. This hormonal ablation presents a grim picture to say the least. Applicants believe that the hormonal stimulation of the sex glands, the adrenals and the pituitary are the result of toxic materials, hormonal derangers and counterfeit hormones, such as, phytosterols produced by the Cyptocides, that upset the balance of the patient's hormones not only by inhibitory effects by production of pseudo or counterfeit hormones that act on the physiologically controlled, normal glands causing abnormal response. Also various kinds of cell poisons and inhibitors destroy the efficacy of the lymphocytes to attack the cancer cells. The cancer cells themselves are prevented from reaching maturation by these cell poisons They are sick cells unable to reach a normal maturity and normal function, whereever they are located and whatever tissue they may be whether glandular, interstital, bone or blood.

The most important thing is to try to destroy the microbes that were producing the aberrant cell inhibitors and false hormones. However, it has been reported that low testosterone levels have been induced in patients with cancer of the prostate by treatment with diethylstilbestrol, a synthetic hormone; and amino-glutethimide, a powerful inhibitor of adrenal corticosteroid biosynthesis, with patient improvement. Furthermore, an immunological mechanism appears to be involved; the inhibition of steroid biosynthesis. By removing the lympholytic effect of corticosteroids, there is produced a marked hyperplasia and increase in the number of circulating lymphocytes which potentiate the immune response. The presence of lymphocytotoxic antibodies has been reported in patients with prostatic cancer. Perhaps this steroid is a "false steroid" and antagonism by the amino-glutethimidine and diethylstilbestrol may permit an increase in the production and circulation of normal lymphocytes capable of attacking the cancer cells.

The role of steroids in chronic diseases was demonstrated by Edward Kendall and Philipp Hench in their studies in rheumatology for which they received the Nobel Prize in 1950. It is true that the steroids do not have an inhibitory effect on these diseases but at the expense of suppressing immunity and permitting the underlying latent infection to continue or to increase in its growth potential.

It is known that a bacterium belonging to the Actinomycetales was able to produce unlimited amounts of steroids from the Mexican yam.

It is stated that some steroids decrease the numbers of circulating lymphocytes as well as blocking immunocompetence. Perhaps the "false steroids" are really responsible for this action. It has been shown that certain toxic antigens prevent the lymphocyte from maturing and become immunocompetent. Leukemia, or an accumulation of large numbers of cells, either lymphocytes or polymorphonuclear leukocytes, may represent a blocking of the pathway to maturity by a toxic agent such as a steroidal or actinomycinlike compound produced by the cryptocides. Perhaps the blocking factor may be related to a protective mechanism directed toward making the cryptocides insusceptible through some biochemical fraction that blocks the immune reaction of the lymphocyte.

The present invention is useful in the treatment of man and/or animal. Safety and effectiveness of the present invention has been demonstrated in animals and has been indicated in the treatment of humans by administration of the aforesaid vaccine form.

Among the various neoplastic diseases (often termed diseases of unknown etiology) subject to palliative treatment are cancer, tumor of the lymphoid tissues, Hodgkin's disease, reticulo-endothelial tissues, arthritis, lymphosarcoma, the broad spectrum of epidermoid cancer, scleroderma, adenocarcinoma, fibrosarcoma, liposarcoma, myosarcoma, acute glomerulonephritis, leimoya sarcoma, osteogenic sarcoma, chondro sarcoma, myeloma, rous chicken sarcoma, coal-tar-induced cancer, fowl leukosis, animal tumors such as Rous, Walker, Sprague-Dawley, Shope and Sarcoma 180, and the like. Many of the foregoing are degenerative and antoimmune diseases.

Malignancy is a neoplastic infection, which depends on the number and virulence of the invading organism, the susceptibility of various organs to it, as well as the natural immunologic components of the host.

The reddish-brown crystalline substances extracted from broths containing organisms within this invention are antibiotic to the bacteria and to all strains of the producing organisms themselves.

MEDIA

The preferred media for growing cryptocides are obtained with Alexander-Jackson's broth, and with Wuerthele-Caspe's autoclaved chick embryo agar. The method of preparation of these two media is given below.

ALEXANDER-JACKSON'S (A-J) SENSITIVE PEPTONE BROTH

Ingredients:
water (distilled)
2,000 ml
beef lung, cut up
2 pounds
peptones 20 grams; 5 grams of each of
(a) myosate, (b) gelysate,
(c) trypticase, (d) phytone
glucose 10 grams
glycerol 80 ml

Boil the beef lung and water for 30 minutes. Filter through cotton or very coarse paper into a flask containing the other ingredients, and heat to dissolve. This crude lung broth can be autoclaved and stored in the icebox, and clarified subsequently. Autoclaving for a second time does not seem to produce any adverse effects.

Clarification:

A 1 to 2 mm layer of infusorial earth (Standard Filter Cel of Johns-Manville Co.) is deposited on a No. 42 Whatman paper disc by laying the disc on a Buchner funnel, applying suction, and then carefully pouring on about 500 ml of a 5 percent suspension of Filter Cel. After the deposition of the layer, when the water goes through clear, the suction flask is well rinsed out. The hot medium can now be filtered through the prepared disc into the flask.

The medium should be filtered a second time through a Buchner-type funnel with a fine fritted glass disc, or else passed once more through the same Filter Cel.

pH adjustment:

The pH of the medium should be adjusted to 7.4 with sodium hydroxide. The medium is then tubed into screw-top glass tubes 150.times.25 mm (Kimble Glass Co., Toledo, Ohio). The tops of the tubes are not screwed tightly until after autoclaving. Autoclave for 15 minutes at 15 pounds pressure. Place about 5 ml of medium into each tube; or place 50 ml in a 250-ml Erlenmeyer flask for primary isolates. Close the flasks with cotton plugs held in a single layer of gauze, and protect the plug by a paper drinking cup or cone.

The A-J broth is obtainable from the Colorado Serum Company of Denver, Colo.

Whole fresh citrated or untreated blood, 0.2 ml is added to 2 ml of broth at pH 7.4, and incubated for a week. A transfer to fresh broth is then made to rid the culture of antibodies or other inhibitory substances. After several days, the organisms appear as a mat at the bottom of the tube. When motile rod forms are present, a soft while ring or pellicle appears. Growth is often seen climbing up the side of the glass tube.

The peptones included in the above broth have been studied individually. Myosate, a pancreatic hydrolysate of heart muscle, favors small virus-like and coccoidal forms. Gelysate, a gelatin hydrolysate, appears to favor slender acid-fast rods and non-acid-fast rods containing acid-fast granules. Phytone a papaic digest of soya meal, and trypticase, a pancreatic digest of casein, both favor the readily growing motile rods. A combination of all four peptones provides a medium which allows a wide variety of forms to develop, and makes it easier to recognize the presence of the organism in primary isolations.

WUERTHELE-CASPE'S CHICK EMBRYO AGAR

The contents of 8-15 days old embryonated hens' eggs are ground up on a Waring blendor, mixed with 1.5 percent melted agar, tubed in screw-top glass tubes, preferably large ones, slanted, an autoclaved at 15 pound for 20 minutes in the slanted position.

Examples of other media which can be used are Difco's brain-heart broth with and without glycerin, Dubos medium, Alexander-Jackson's modification of von Szabocky's glycerol lung broth, dextrose blood agar, Alexander-Jackson's adaptation of Bushnell's poi agar, Petragnani, Lowenstein-Jensen, Dorset egg media, Witte's peptone, Difco's bactopeptone, Armour's peptone, a Merck peptone and Fairchild's peptone.

METHODS OF IDENTIFYING CRYPTOCIDES

This invention also involves a test that will show the existance of a neoplastic disease before the tumor exists, or the existance of a chronic underlying infection in man and/or animal. Until now any aberrant symptom of a patient has to be evaluated in the light of a latent cancer until it was ruled out. A fever of unknown origin could turn out to be a sarcoma somewhere in the body made manifest weeks later after much laboratory work and X-rays. By then, it was already too late, to do anything. Even if it had been known that cancer was imminent there was no treatment. There was nothing to do but wait until a tumor presented itself and then attempt to cut it out or destroy it by radiation or chemicals. (Applicants' invention involves a cure for such cancer.)

Tests for determining the presence of cancer such as the Pap smears testshave serious problems associated therewith. In the Pap smears tests, the body cells that are cast off from the uterus, cervix and vagina are smeared from the cervix, are placed on a silde and stained. Not only is the presence of cancer cells detected but the amount of estrogen in the body is indicated by the size and shape of the nucleus of the cell in relation to the cytoplasm. This test is useful in determining the stage of menopause in women. Unfortunately, when the smear for cancer is positive, the cancer is already there. However, it does permit early detection of some kinds of cancer of the female reproductive organs. The same method of cell determination is now applied to a number of other sites such as lung and stomach.

As cancer is an infection, surgery, radiation and chemicals cannot eradicate a continuing infectuous process. For example, cobalt machines may reduce the size of tumors but contribute very little to the long-term cure of the disease.

The test of this invention allows a screening program of the entire poputation by means of routine blood cultures to determine the presence of the cryptocides bacteria correlated with evaluation of blood smears and related to immune competency by various methods of antigen-antibody determination.

There are a number of identifying biochemical tests that can be applied but these are too time-consuming and expensive for a routine laboratory. In the dying patient, a few drops of blood taken from the antecubital vein of the arm will grow out furiously on direct plating on the solid blood plates. Usually, isolation from blood is done by placing ony a few drops of blood, about five, in the bottom of a peptone-broth tube, and incubating. The organisms can be readily recognized either in hanging drops of the living cultures or by appropriate staining. The organisms grow up the side of the tube forming a lacy pattern and then produce a pellicle or doily on the surface. These usually signify the presence of motile rods. This is a good stage from which to make a vaccine. As the pellicle ages it has a tendency to drop into the tube again the spore stages are then formed. The spores cannot be used for vaccines as it is almost impossible to kill them. The liquid cultures will often transfer to solid media plates. Applicants' sensitive peptone broth for primary isolation is useful, and it can be obtained from the Colorado Serum Company in Denver, Colo. Dr. Diller's paper gives the various methods of isolation using the technique of Von Brehmer, Glover, Seibert and others. Applicants have also used synthetic broh media for primary isolation but these proved to be too toxic on animal experimentation.

There are several other ways of making primary isolations of the cryptocides. Sterilely obtained tumor tissue fresh from the operating room can be placed into liquid media and later transferred to solid blood plates. Some people have ground up the tumors, filtered them and then cultured them. This is difficult because of the problem of maintaining sterility. These methods led to the recovery of the specific microorganism, the cryptocides. Still others have made various extractives of the tumors with alcohol, acetone or other solvents and used these for the vaccine. Another method is to grow the organism from one of its favored spots, the roots of infected teeth or tonsils. However, the mouth contaminants must be eliminated. Still another way is to dilute the patient's blood with equal parts of distilled water in order to disrupt the red cells wherein the parasites are contained as well as in the serum. The tubes are lightly boiled over an open flame two or three times and then incubated for eight to twelve days. Intervening examination of the blood will reveal the rate of growth. When the growth is abundant, usually in ten to twelve days, the blood can be filtered to remove larger particles, then formalinized, standardized and tested for viability. This method may have some advantages over the whole-cell antigens obtained by the Crofton method, because the whole-blood cultures will also contain toxins and antitoxins as well as many of the minute forms which do not grow out on artificial media. This is the German method.

The following is a description of applicants' test method for the quick detection of the presence of the chorionic gonadotropic hormone (which is produced by cryptocides) in urine which indicates the existence of a chronic underlying infection in tissue and blood which produces the chorionic gonadotropic hormone, thus eliminating the need for study of individual colonies. This test can be of great importance to the medical profession, particularily in determining the presence of cancer or the likelihood of cancer proliferation. The test can be used in man and animal.

Applicants theorize that the abnormal production of the steroid chorionic gonadotropic hormone, keeps the cancer cells growing. Chorionic gonadotropic hormone is made by the microbe Cryptocides.

This tool allows the diagnosis and prognosis of chronic underlying infection or condition. Treatment can follow.

Describing the diagnoistic test, the way that applicants have found to indicate whether or not choronic gonadotropic hormone is present in the urine is: to take a predetermined amount (usually 10 ml.) from a urine specimen of the person or animal; prepare a blood culture using the urine sample; allow the blood culture to set (e.g., one day); take a predetermined amount (e.g., 3 to 5 drops) from the blood culture and directly place it in the peptone broth described elsewhere herein; and incubate (e.g., at 37 DEG C. for 24 hours) the peptone broth. Blood plasma or serum can be used in a suitable container such as a test tube. Blood is its own media and no media need be added to it. This test procedure will give a negative or positive result depending upon the absence or presence, respectively, of chorionic gonadotropic hormone in the urine speciment. (It should be noted that minimal essentially, undetectable amounts of chorionic gonadotropic hormone may be present in the urine due to the presence of pathologically insignificant amounts of Cryptocides that are universally present in man and animal.) A positive indication (colony growth) will occur, if it is going to at all, in approximately ten days.

(The microorganism from the colonies from the urine-blood cultures may be tumorogenic and antibiotic.)

Negative, e.g., hormone, indications are always obtained when the sample from the blood specimen is first cultivated on a plate (e.g., agar) and then the colony is placed in the peptone broth. Negative indications are always obtained when the urine is culture on a blood plate (i.e., media has been added) and the colony is then placed (e.g., place blood plate with colony between fingers and squeeze over top of the broth test tube) in the peptone broth. In both instances negative indications are present even when more than normal amounts of chorionic gonedotropic hormone are present in the urine. Proper postivie indications are obtained when blood in test tube is used, but improper negative indications are obtained when blood in plates using a solid media is used. It has been reported that Cryptocides is anaerobic, which may explain the aforegoing. Also there may be present a mixture of phases of the microbe due to the pleomorphic nature of microorganism.

A positive indication means that the person or animal from whom the urine specimen was taken has a chronic underlying disease or infection, such as, cancer. This test can be used to detect all of the above listed neoplastic diseases.

Describing the prognostic test, the same procedure is used as described above for the preparation of the incubated peptone broth. Any resultant positive indication is measured conventionally for intensity or magnitude and/or length of duration of such positive indication. This gives results or measurements that can be compared with prior or future results or measurements from other incubated peptone broths made from the same patient. The prognostic test gives qualitative and quantitative results.

Since the presence of the bacterium can be proven by the product it makes, if the patient is well, the bacterium is attenuated (surpressed) and does not make significant amounts of the product. When the patient gets worse, cryptocides makes more chorionic gonadotropic hormone so prognostic tests can successively be made.

Corroboration was achieved by passing cultures, having positive indications, from primary blood isolates into the next test tube with the presence of blood. The passed cultures were allowed to grow until visible growth was seen. The growth was tested and it gave positive indications.

Another test for determining or detecting the presence of chorionic gonadotropic hormone which indicates the presence of a chronic underlying disease or infection in blood and tissue which produces the chorionic gonedotropin hormone involves a skin test. The vaccine described above is subcutaneously placed under the skin. If a welt appears, it is a positive indication that chorionic gonadotropin hormone is present and that a chronic underlying disease or infection exists in the patient.

A variation of the above skin test involves conventionally extracting a tuberculin-like substance from the vaccine described above, and subcutaneously inserting the extracted substance. If a welt appears, it is a positive indication that chronic gonadotropic hormone is present and that a chronic underlying disease or infection exists in the patient.

The urine test is the preferred detection test.

Applicants' urine test is exquisitively sensitive to a chorionic gonadotropic hormone--there are no interferring substance In other words, applicants' urine test is specific to chorionic gonadotropic hormones--it is a serological, higly specific test.

The urine speciments from seven cancer patient were each placed into separate test tubes containing only A-J peptone broth as the medium. There was specific growth in each test tube. The colonies were removed in each instance and were subjected to separation (extraction) processes until only the microorganism was left. Each of the seven isolated microorganism specimens were placed in separate test tubes containing only A-J peptone broth (no blood or agar was used). In each of the seven tubes, a positive reaction was obtained confirming the fact that the microorganism which produces chorionic gonadotropic hormone was present.

Applicants do not known of any other microorganisms besides Cryptocides which produces chorionic gonadotropic hormone, which gives a positive result in applicants' urine test.

It is known that the standard pregnancy test will give a positive indication when chorionic gonadotropic hormone is present. It is also known that a positive indication can be obtained in the pregancy test from aspirin.

Some state that cancer is essentially a cesspool for the collection of microorganisms, but applicants have found that only Cryptocides causes cancer and it is the only microorganism which produces chorionic gonadotropic hormone. Dr. Ross and others have stated in several instances that chorionic gonadotropic hormones are produced when a patient has cancer and that the chorionic gonadotropic hormone emanates from cancer.

Applicants have found that the chorionic gonadotropic hormone and/or biologically related hormones and sterols which yield a positive indication in a standard pregnency test and which can be identified by gas chromatography emanates from Cryptocides, which causes the cancer. An example of such a biologically related sterol are the phytosterols. An example of such a pregnency test is Walpole's "UCG-Test" pregnancy test which detects human chorionic gonadotropic immunologically in the urine of pregnant women.

Dr. Ross's tests used blood and found the presence of chorionic gonadotropic hormone. These are called the L-tests. Applicants are able to detect cancer by detecting the presence of chorionic gonadotropic hormone in urine. Applicants' can use their test in a diagnostic sense, that is, it can be used to determine if cancer is present. Applicants' can also use their urine test in a prognostic sense, that is, they can use it to see whether or not the amount of chorionic gonadotropic hormone has increased or decreased, and can use it to indicate the treatment to be used (depending on whether the patient's condition is better or worse).

Lactrile states that the chorionic gonadotropic hormone which is in the embryo stage is different from the other stages. Applicants' urine test detects chorionic gonadotropic hormone regardless of the stage it is in. (Kitts stated that all of the cancer cells are in the first stage).

Chorionic gonadotropic hormone is a water soluble, gonadstimulating glycoprotein.

VACCINE PREPARATION

Researchers for a number of decades have been seeking tumor antagonists not only in the form of antibiotics, which are chemicals secreted by other microorganisms, but through potential immunization by the use of other microorganisms. These biologicals are in sharp contrast to the chemotherapeutic agents that seek to destroy the dividing tumor cell regardless of the entailed immune suppress Applicants have used the causative agent itself as a menas of immunization.

Applicants have prepared and used an autogenous vaccine for the treatment of chronic, ongoing infections. Customarily the vaccines are prepared from urine, nasal, throat and bowel secretion as well as from various tissues and other secretions. The vaccines are used for the building up of immunity in the chronically ill patient who suffers from a failure to produce immune bodies against his chronic infection. This state of nonresponse is called immunoincompetence. Applicants do not represent to the cancer patient that the use of autogenous vaccine is proposed for the treatment of cancer but for their underlying failure of immune competence. In many cancer patients applicants do not use autogenous vaccine. The use of vaccines must be carefully weighed in the evaluation of the patients' immune status. In some cases the use of vaccines are actually contraindicated. In the seriously ill cancer patient the most important thing is to raise the patient's immunity by the use of fresh whole blood transfusions from suitable donors, and by the use of antibodies such as gamma globulin. The next most important thing is to treat their chronic underlying infection whatever it may be with suitable antibiotics. The removalof harmful substances from the diet is essential as well as the addition of needed vitamins and nutriments that may be lacking in the seriously ill because of lack of appetite and weight loss and faulty diet. Applicants do not believe that vaccines can cure the cancer patient. It is one of the modalities used for the chronically ill whatever their disease in the effort to restore their resistance to an ongoing; underlying chronic disease.

It is now incontrovertible that the cancer disease results in the loss of immunity yet it is treated with radiation which destroys immunity and with drugs which encourage cancerous growth. An abstract of "Carcinogenicity Studies of Clinically used Anticancer Agents", D. P. Griswold, J. D. Prejean, A. E. Casey, J. H. Weisburger, E. K. Weisburger, H. B. Wood, Jr., and H. L. Falk. Southern Research Institute, Memorial Institute of Pathology and Baptist Medical Center, Birmingham, Ala. 35205, and National Institutes of Health, Bethesda, Md. 20014, shows a number of drugs, which produce cancer in experimental animals, yet whose use is advocated by a government agency and the medical profession. Such drugs include Melphalan, Chlorambucil, uracil mustard, Natulan, dimethyltriazenoimidazole carboxamide, and 1,3-bis (2-chloroethyl-) and 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea. Some of these are so-called anticancer drugs. Tumors of several types and at a variety of sites were seen in mice and rats during a testing of such drugs for carcinogencity.

Applicants repeat that they do not treat cancer with vaccines, use autogenous vaccines obtained from the patients' own tissues and body fluids to treat an underlying chronic infection. These organisms are not present in cancer alone but also in a host of collagen diseases and in healthy carriers as well, and that the use of vaccines for chronic disease states is an accepted modality in therepeutic medicine. Use of applicants' autogenic vaccine is therefor beneficial to a patient whether or not the patient has cancer. However, by giving the patients good nutrition, by helping them fight off chronic infection, and by using any and all accepted modalities that may be helpful, applicants assist the patients in throwing off their diseased condition, whatever it may be.

The production of vaccines from blood cultures is a rather long and tedious procedure. Applicants use the following method for making autogenous vaccines from urine. Applicants use sheep cell blood agar with phenyl ethyl alcohol, which inhibits the growth of E. coli, a common contaminant. Either the patient leaves the urine as directed with a testor or follows these directions at home:
1. Boil a screw-top bottle and top for twenty minutes. Let it cool. Remove with sterile tongs which have been boiled or disinfected in rubbing alcohol.
2. Take a bath. If a female, take a douche and wash off thoroughly.
3. Using three balls of sterile cotton wash off from front to back three times over the perineum with either Phisohex or some other mild disinfectant such as St 37. If a male, pull back the foreskin and wash throughly three times separately with each of the three cotton balls.
4. Start the urine stream over the toilet bowl and then catch the midstream into the sterile bottle without contaminating the inside. Be careful to keep the fingers out of the inside of the screwtop cap. Tighten the cap throughly to prevent leakage.

When the urine is received in the laboratory it is streaked onto the sterile blood plates using sterile swabs. The plates are then incubated in the usual way. Generally within 12 hours small colonies have formed. When these have been properly identified by allowing the growth to continue for a day or two to be sure that the characteristic colonies are present, then a single colony is studied by Ziehl-Neelsen stain, Kinyoun type, for acid fastness and the characteristic morphology. Then a single, identified colony is spread on one or two additional plates where they are incubated until sufficient growth has accurred. Stained preparations are again examined. The colonies are then swabbed off into a 2 percent phenol solution and permitted to stand overnight. Then after about eight hours, the phenolized cultures are diluted to 0.5 percent phenol. It requires about two weeks to complete the sterility tests and to make several dilutions according to government regulations. Autogenous vaccines tailored for each individual are prepared, but this procedure is not so limited. The vaccines are made up into 10 million, 100 million, and 1,000 million (=1 billion) organisms per c.c. The lowest amount, 10 million organisms per cc, is used as the starting bottle for progressive immunization. Doses are taken every three to five days depending on the reaction. It is wise to start with 0.1 cc by subcutaneous injection of the lowest amount and observe for evidence of redness or soreness at the site of the injection or symptoms of hpersensitivity such as mild fever, lalaise, or muscle or joint pains. if there is a mild reaction, the patient waits until it subsides. before repeating the same dose or smaller in three to five days by mouth. If there is no reaction, then the dose is increased by 0.1 cc to 0.2 cc and administered first by subcutaneous and then orally in three to five days. The third week, the dose is increased again by 0.1 cc to 0.3 cc subcutaneously and repeated orally again in three to five days. The oral dose is taken under the tongue and held in the mouth for absorption. The vaccine is increased in this manner until twenty drops are taken. Then the next higher dilution of organisms is started in bottle number two or 100 million organisms per cc. The starting dose is only 0.1 cc since the second dilution is ten times as strong as the first one. Again the doses are increased gradually and so on with the other bottles of the vaccines. A vaccine usually lasts six months but if there is quite a change in the character of the organisms under treatment the occasionally it is good to prepare a new vaccine in three months. This method just described is applicants' preferred method of preparation of autogenous vaccines and their administration.

The single most important factor in the presentation of vaccines is to rule out common contaminents. The colonies can be entirely confluent in severely infected hosts so that a transplant must be made in order to isolate individual colonies for study. The typical colony has an umbonate (fried egg) shape and may or may not be hemolytic. The colonies may also be wrinkled or smooth, china white or pale tan and even pale pink or orange when grown in the dark. The slides are made by lightly wiping a culture from the plate with a sterile cotton swab or with a platinum loop onto the surface of the glass slide and fixing it with gentle heat. One colony only should be selected and a cross-section should be studied by taking samples from the center outward to the periphery to obtain the different pleomorphic stages. If the material is handled gently they ray formation of the growth will not be broken up. The Kinyoum modification of the Ziehl-Neelsen stain is used since it can be applied in the cold for five minutes and does not require heating. The red dye is washed off with sterile distilled water and the slide is then briefly decolorized with 1 percent hydrochloric acid in 70 percent alcohol. The Cryptocides organisms are more sensitive to decolorization by acid-alcohol than the tubercle bacillus. The slide is washed again and the methylene blue counterstain is applied, 6 to 8 drops of normal (4%) of sodium hydroxide are added. After 30 seconds it is washed off. After the slide is air-dried it is ready for examination under the light microscope at not less than .times.800 with oil immersion. If slides are prepared from tissue impression smears of tumors, the same procedure is followed but Alexander-Jackson's triple stain may be applied to duplicate slides in order to differentiate the non-acid fast forms of Cryptocides from common contaminants. At times, the Cryptocides organism is not acid-fast in some stages of its growth.

Usually the organism isolates out in the coccal form which has led many investigators to believe they are dealing with a staphylococcus. However, the cocci will be both acid-fast and non-acid-fast and will vary greatly in shape from the very small to the large globoidal or sac forms which often stain blue and appear to be spilling out the red acid-fast cocci much as marbles out of a bag. In addition, the cocci appear to split longitudinally into small rods. The cocci, after a period of time, have small filaments spouting from them which turn into rods that are red or acid-fast. If the culture is not mutilated by rough handling, often the large tublar forms can be seen which are observed by darkfield microscope in fresh blood. These are very delicate and disrupt easily. The ray formation may also be apparent but the sheath is extremely diaphanous and is destroyed often in the staining process. Sometimes the cancer organism can isolate out primarily as a rod or even as a branching hyphal form. At other times clublike bodies are seen which are blue in color and contain the acid-fast bodies within them. It is very important to study a number of the colonies on the plates and to be sure that the various transitional forms of the organism can be seen in one isolated colony. Only then may the organism be grown in sufficient amount to harvest for the vaccine. All of this work requires careful examination and experience to be sure of the growth pattern and morphology of the Cryptocides.

The organisms isolated from the urine cultures have been classified under various names such as staphylococcus epidermidis and enterococcus fecalis, in other words, common organisms found on the skin and in the bowel. However, by careful sterile methods microorganisms are found to be growing from the urine in great abundance. Microbiologists are still debating the nature and classification of these organisms. A recent paper, which appeared in Transactions of the New York Academy of Sciences, by Dr. Florence Seibert, claims that these isolates from her material which yield certain supposedly well-known microorganisms are not the standard well-recognized types at all but the acid-fast organisms which we have classified as the Cryptocides. Microbiologists who examined applicants' urine cultures state that there are a variety of organisms in applicants' urine vaccines. What is known is that these organisms occur in large numbers and are often hemolytic (destructive of red blood cells). Very possibly urine cultures contain a mixture of whatever microbes that happen to filter through the kidneys from distant body foci. Applicants use the mixture in vaccines only as nonspecific immune booster in chronic disease.

The vaccine can be prepared by the following preferred method. To 10 cc of heparinized freshly-drawn sterile blood add 10 cc sterile distilled water. Heat over the Bunsen burner to boiling several times (to break the red cells). Incubate for 10 to 12 days at 37 DEG to 38 DEG C. This gives the growing culture without addition of media, since blood acts as its own medium without the addition of anything else. Add 4 percent formalin (formaldehyde) to inactivate (kill) the P. Cryptocides. The admixture is put through a microfilter to remove the dead or microorganisms. The live attenuated microorganisms go through the filters. Then dilute the filtrate with sterile saline solution until the final solution contains 1% formalin. This is then tested for sterility and than can be used as an autogenous vaccine.

The autogenous vaccine can be used to immunize against underlying chronic diseases or infections, or neoplastic diseased which produce choroninic gonedotropic hormone and/or biologically related hormones and sterols which yield a positive indication in a standard pregnancy test and which can be identified by gas chromatography and which are caused by Cryptocides.

EXAMPLE

Materials and Methods

Female mice of strain A/He, 2 months old, were divided into 4 groups of 16 animals each. The average body weight for each group was 28.1 g. Animals all received 12 intrapertoneal injections, 3 injections per week for 4 weeks (Mondays, Wednesdays and Fridays). Sterile disposable plastic syringes fitted with 25-gauge needles were used for each injection.

The mice were housed in plastic shoe box-type cages, 8 per cage, and fed a standard Teklad mouse diet and water ad libitum. They were weighed before each injection and then weekly following the injection period. The injections contain the reddish-brown crystalline material extracted from broth containing organisims within the scope of this invention.

Dosages used Dose/Injection
N.C. var. 0.13 mg/0.1 ml
Control 0.13 mg/0.1 ml
#6 1.40 mg/0.1 ml
#5 0.28 mg/0.1 ml

At 20 weeks after the last injection, the mice were killed by cervical dislocation. Necropsies were performed and the lungs removed and fixed in Tellyesniczky's fixitive for 24 hours. The number of tumor nodules on each lung was determined after fixation by counting with the naked eye.

The animals tolerated the materials extremely well and no adverse effects were encountered.

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1479838/
J Transl Med. 2006; 4: 14.

Bacteria and cancer: cause, coincidence or cure? A review

DL Mager

Abstract

Research has found that certain bacteria are associated with human cancers. Their role, however, is still unclear. Convincing evidence links some species to carcinogenesis while others appear promising in the diagnosis, prevention or treatment of cancers. The complex relationship between bacteria and humans is demonstrated by Helicobacter pylori and Salmonella typhi infections. Research has shown that H. pylori can cause gastric cancer or MALT lymphoma in some individuals. In contrast, exposure to H. pylori appears to reduce the risk of esophageal cancer in others. Salmonella typhi infection has been associated with the development of gallbladder cancer; however S. typhi is a promising carrier of therapeutic agents for melanoma, colon and bladder cancers. Thus bacterial species and their roles in particular cancers appear to differ among different individuals. Many species, however, share an important characteristic: highly site-specific colonization. This critical factor may lead to the development of non-invasive diagnostic tests, innovative treatments and cancer vaccines.

Introduction

An overwhelming body of evidence has determined that relationships among certain bacteria and cancers exist. The bacterial mechanisms involved are as yet unclear. These gaps in knowledge make it impossible to state the exact progression of events by which specific bacteria may cause, colonize or cure cancer. Therefore, many questions remain. For example, why do infections that are wide spread appear to cause cancer in only a minority of individuals? Do certain infective agents initiate or promote cancer or does an early undetected cancer facilitate the acquisition of the infection? Can the exposure to or colonization of specific bacteria prevent or treat certain cancers? Can the highly site specific colonization of certain bacteria for a tumor be clinically useful in the diagnosis of cancer or delivery of a therapeutic agent?

The scope of this review is broad therefore a wide range of reports is presented. Recent findings that have found associations between certain bacterial infections and tumor development will be discussed as well as genetic factors that may predispose individuals to "cancer- causing" infections. Mechanisms thought to be involved with the carcinogenic, diagnostic and preventive or treatment roles of bacteria are introduced. As the carcinogenic potential of viral agents and H. pylori has been reviewed extensively elsewhere, it will not be included here.

Bacteria and carcinogenesis

It is estimated that over 15% of malignancies worldwide can be attributed to infections or about 1.2 million cases per year. Pisani et al. [1] Infections involving viruses, bacteria and schistosomes have been linked to higher risks of malignancy. Although viral infections have been strongly associated with cancers [2,3] bacterial associations are significant. For example, convincing evidence has linked Helicobacter pylori with both gastric cancer and mucosa-associated lymphoid tissue (MALT) lymphoma [4-6], however other species associated with cancers include: Salmonella typhi and gallbladder cancer [7-10], Streptococcus bovis and colon cancer [11-14] and Chlamydia pneumoniae with lung cancer [15-17]. Important mechanisms by which bacterial agents may induce carcinogenesis include chronic infection, immune evasion and immune suppression [18].

It has been shown that several bacteria can cause chronic infections or produce toxins that disturb the cell cycle resulting in altered cell growth [15,16,19]. The resulting damage to DNA is similar to that caused by carcinogenic agents as the genes that are altered control normal cell division and apoptosis [20,21]. Processes that encourage the loss of cellular control may be tumor initiators (directly causing mutations) or promoters (facilitating mutations). Tumorigenesis is initiated when cells are freed from growth restraints, later promotion results when the immune system is evaded favoring further mutations and increased loss of cell control. As the tumor proliferates an increased blood supply is needed resulting in the organization of blood vessels or angiogenesis. Subsequent invasion occurs if the tumor breaks down surrounding tissues. The worst outcome is metastasis which results when cells break away from the tumor and seed tumors at distant sites [8].

The immune system is an important line of defense for tumor formation of malignancies that express unique antigens. Certain bacterial infections may evade the immune system or stimulate immune responses that contribute to carcinogenic changes through the stimulatory and mutagenic effects of cytokines released by inflammatory cells. These include reactive oxygen species (ROS), [22,23], interleukin-8 (IL-8) [11], cyclooxygenase-2 (COX-2), [24], reactive oxygen species (ROS) and nitric oxide (NO) [25]. Chronic stimulation of these substances along with environmental factors such as smoking, or a susceptible host appears to contribute significantly to carcinogenesis.

Salmonella typhi and gallbladder cancer

Worldwide annual incidence of gallbladder cancer (GC) is 17 million cases with high incidence rates in certain populations. The malignancy is usually associated with gallstone disease, late diagnosis, unsatisfactory treatment, and poor prognosis. The five-year survival rate is approximately 32 percent for lesions confined to the gallbladder mucosa and one-year survival rate of 10 percent for more advanced stages [26]. Over 90 percent of gallbladder carcinomas are adenocarcinoma [27] involving gallstones in 78% – 85% of cases [26].

There are several risk factors for gallbladder cancer. The main associated risk factors include cholelithiasis (especially untreated chronic symptomatic gallstones), obesity, reproductive factors, environmental exposure to certain chemicals, congenital developmental abnormalities of the pancreatic bile-duct junction and chronic infections of the gallbladder [26,28]. The interplay of genetic susceptibility, lifestyle factors and infections in gallbladder carcinogenesis is still poorly understood [29], however a link has been specifically proposed between chronic bacterial infections of the gallbladder and Salmonella typhi [26].

The strongest epidemiological evidence of bacterial oncogenic potential, aside of Helicobacter pylori, concerns S. typhi. Infection with this bacterium of typhoid, can lead to chronic bacterial carriage in the gallbladder [30]. Recent epidemiological studies have shown that those who become carriers of S. typhi have 8.47 times the increased risk of developing carcinoma of the gallbladder compared with people who have had acute typhoid and have cleared the infection [26]. These findings agreed with earlier investigations by Welton et al. [31] and Caygill [30].

A case-control study by Welton et al [31] compared those who experienced acute infection with S. typhi to those who subsequently became chronic carriers following the 1922 typhoid outbreak in New York. Carriers were six times more likely to die of hepatobiliary carcinoma than matched controls. Additional evidence was found in an analysis of the 1964 typhoid outbreak in Aberdeen [30]. Their findings also suggested a strong association between chronic carrier status and hepatobiliary carcinoma. These studies also agreed, people who contracted typhoid but did not become carriers were not at higher risk of cancer [8,26,30,31].

The highest incidence of gallbladder cancer (GC) in the world is among populations of the Andean area, North American Indians, and Mexican Americans. In Europe, the highest rates are found in Poland, the Czech Republic and Slovakia. The high rates observed in Latin America are primarily in populations with high levels of Indian mixture [32]. This evidence supports the notion that increased susceptibility to gallbladder cancer depends on genetic factors that predispose people to gallbladder cancer either as primary factors, or secondarily as promoters by favoring the development of cholesterol gallstones. The highest mortality rates are in South America, (3.5–15.5 per 100,000) and among Mexican Americans [26]. Incidence rates of GC in various ethnic groups in the USA confirmed the worldwide pattern, as GC was substantially more frequent among Hispanic than non-Hispanic white women and men. Interestingly, compared to non-Hispanic whites an excess of GC was also reported among American Indians in New Mexico, in agreement with the excess in incidence rates reported for American Indians and Alaskan Natives [33]. The malignancy is 3 times higher, however, among women than men in all populations [26].

Two main pathways to GC exist worldwide. The predominant pathway involves gallstones and resultant cholecystitis and affects women to a greater extent than men. The risk of developing gallstones in response to environmental factors is genetically determined, as shown by the marked tendency of gallstones to cluster in families [34]. The other pathway involves an anomalous pancreatobiliary duct junction (APBDJ), a congenital malformation of the biliary tract that is more frequent in Japan, Korea, and possibly China, than in Western countries [28]. In APBDJ, the premature junction of common bile and pancreatic ducts results in regurgitation of pancreatic juice into the gallbladder, leading to bile stasis and inflammation, though generally less severe than that resulting from gallstones [28].

Currently the prevention of gallbladder cancer in high risk populations depends upon the diagnosis of gallstones and removal of the gallbladder. Indeed, a strong inverse association between number of cholecystectomies and GC incidence and mortality rates can be found in many countries. The increase of GC mortality reported in Chile in the 1980s was related to decreased rates of cholecystectomies [35]. Increased rates led to the removal of gallbladders at risk, and a reduction of GC incidence and mortality in Europe and the United States [36].

Unfortunately, information about the genetic changes involved in gallbladder carcinogenesis is limited. Most studies have focused on gene abnormalities and deletions ("loss of heterozygosity") at chromosomal regions harboring known or putative tumor suppressor genes [28]. It appears, however, that TP53 inactivation has an important and early role in gallbladder carcinoma associated with gallstones and chronic inflammation. This inactivation would abrogate the tumor suppressor function of the p53 protein resulting in impairments in cell cycle control, cellular repair and apoptosis.

In contrast, KRAS mutations are frequent and early events in tumors associated with APBDJ [28] but detected less often in gallbladder carcinomas associated with gallstones. KRAS is an oncogene that encodes a protein that is a member of the small GTPase family. A mutation in this gene results in an abnormal protein implicated in several malignancies, including lung adenocarcinoma, ductal carcinoma of the pancreas and colorectal carcinoma among others.

Chlamydophila pneumoniae and lung cancer

Lung cancer is the leading cause of cancer death in the United States and many countries in the Western world. In 2002, the most recent year for which statistics are available, 90,121 males and 67,509 females died from lung cancer [37]. About 6 out of 10 people with lung cancer die within 1 year of finding out they have lung cancer. Between 7 and 8 will die within 2 years [38]. Although patients may experience a partial or complete response to treatment, most patients relapse and die. Increased dosage of chemotherapy or length of treatment has not been beneficial [39].

Chlamydophila (formerly Chlamydia) pneumoniae infection has been implicated in several chronic lung diseases by serology and direct antigen detection. Acute lower respiratory tract infection caused by C. pneumoniae seems often to precede attacks of asthma in both children and adults but is also involved in some exacerbations of chronic bronchitis. More importantly it seems to be strongly associated with chronic obstructive lung disease irrespective of exacerbation status. Moreover, persistently elevated C. pneumoniae antibody titers have been observed in sarcoidosis and lung cancer [40].

C. pneumoniae is a Gram-negative bacillus and an intracellular parasite that causes respiratory infection in more than 50% of adults. The route of transmission is usually by aerosol and in most cases these infections are mild. The bacterium is, however, an important cause of pneumonia, bronchitis, sinusitis, rhinitis and chronic obstructive pulmonary disease [41]. Respiratory infections from C. pneumoniae vary in different countries and populations, being endemic in the United States and epidemic in Scandinavian countries [19].

After acute infection the C. pneumoniae intracellular life cycle is characterized by the development of metabolically inert (and thus antibiotic resistant) atypical "persistent" inclusions. These inclusions contain increased quantities of chlamydial heat shock protein 60, a highly immunogenic protein implicated in the pathogenesis of chronic chlamydial infections. The resulting clinical course is acute symptomatic illness followed by chronic respiratory symptoms. Research also suggests that persistent C. pneumoniae inflammation correlates with increased risk of lung cancer [16,17,19]. Prospective and retrospective studies both report that individuals with elevated IgA antibody titers to this organism have 50% to 100% increased lung cancer risk [15].

In a study by Kocazeybek et al. [19] the relationship between chronic C. pneumoniae infection and lung carcinoma was examined. A total of 123 patients who were smokers and diagnosed with lung carcinoma based on clinical and laboratory (radiological, cytological) findings were examined. 101 (82.1%) of the cases were male. 70 had small-cell, 28 squamous-cell and 7 large-cell carcinomas, while 18 had adenocarcinoma. 123 healthy controls were matched to the cancer patients by age, gender, duration of smoking and locality.

Blood samples (5 ml) were withdrawn at the time of diagnosis (or enrollment for controls) and 1 month later. Values between IgG ≥512 and IgA ≥40 were set as the criteria for chronic C. pneumoniae infections. In male patients with lung carcinoma, IgG antibody titers of ≥512 and IgA antibody titers of ≥40 were found at a higher rate than in the control group, however, this ratio was not significant for female patients. These elevations in antibody titers were found in a total of 62 (50.4 %) cases, 54% of the male patients and 36% of the female patients. Chronic C. pneumoniae infections were seen statistically more often in male patients with carcinoma who were aged 55 years or younger than in controls (P < 0.001). No difference was reported between male patients with lung carcinoma over age 55 and controls or in blood titers between female patients and controls.

The relationship between C. pneumoniae infection and lung carcinoma was studied by Littman et al. [42] in a large prospective case-control study to investigate whether IgA antibody titers to C. pneumoniae were associated with lung cancer risk. A total of 508 pairs were enrolled and included both current and former smokers. Serum was collected at baseline and annually thereafter. Antibody determinations of each lung cancer subject and matched control were tested simultaneously in the same titration series in a blinded fashion. C. pneumoniae titers (IgA or IgG) ≥16 were considered seropositive, which was consistent with the cutoff used in other studies. Subjects were matched by age, gender, and smoking status at baseline. The median age of cases and controls was 59 years and about half were women. All subjects were also examined for demographic, lifestyle, dietary, and racial and ethnic factors. Lung cancer subjects had a heavier smoking history than controls.

After adjusting for a history of chronic bronchitis or emphysema, lung cancer subjects were more likely to have IgA titers ≥16 (55.4% vs. 51.3%) and ≥256 (5.1% vs. 2.5%) to C. pneumoniae than controls. Individuals with antibody tiers IgA ≥16 had 1.2 times the risk of lung cancer (95% confidence interval, 0.9–1.6) compared to those with lower titers. Investigators reported a significant trend (P = 0.007) of increasing odds ratios with increasing IgA titers primarily due to an odds ratio of 2.8 (95% confidence interval, 1.1–6.7) associated with titers ≥256. Elevated IgA was reported with squamous cell carcinomas and to a lesser extent, for small cell carcinomas and adenocarcinomas. There was no evidence of a stronger association with elevated IgG titers however. Subjects with race not classified as White or Black were more likely to have IgA titers ≥16. No significant differences in seropositivity were found, however, based on smoking behaviors.

Streptococcus bovis and colorectal cancer

Colorectal cancer (CRC) is a common malignancy in developed countries and is the 3rd most common cancer in the United States [38]. Greater than 80% occur sporadically [43]. The American Cancer Society estimates that there will be about 104,950 new cases of colon cancer and 40,340 new cases of rectal cancer in 2005 in the United States. Combined, they will cause about 56,290 deaths. The risk of colon cancer increases after the age of 40 and rises exponentially from the ages of 50 to 55. In fact, more than 9 out of 10 people found to have colorectal cancer are older than 50 [38].

Survival of CRC is related to the stage of disease at the time of the initial diagnosis. Between 1985 and 1997, death rates of colon cancer in the United States declined slightly due to earlier detection of primary tumors, via stool blood tests, sigmoidoscopy, colonoscopy, and screening tests for serum carcinoembryonic antigen concentration (CEA) [44]. The 5-year survival rate for CRC patients is greater than 90% when tumors are detected at a localized early stage. After the cancer has spread regionally and involves adjacent organs or lymph nodes, the rate drops to 40–65%; survival is less than 10% for patients with distant metastases. Therefore, there is an urgent need to develop effective treatment strategies to reduce morbidity and mortality. Surgery is currently the primary treatment modality for this disease. By the time the patient presents with recurrent symptoms, however, the disease is rarely curable by surgery even when combined with other therapies [45].

Several species of bacteria have been linked to chronic infections of the colon and increased risk of colon cancer including Escherichia coli [46] and several streptococci [47,48]. Recent studies, however, have validated earlier findings of an association between colon cancer and Streptococcus bovis [11,12]. As early as 1951, McCoy and Mason [49] suggested a relationship between colonic carcinoma and the presence of infectious endocarditis. It was not until 1974 [50] that the association of Streptococcus bovis and colonic neoplasia was recognized, as 25–80% of patients who presented with a S. bovis bacteremia had a colorectal tumor. The incidence of S. bovis associated colon cancer has been determined as 18% to 62% [14].

S. bovis is a normal inhabitant of the human gastrointestinal tract that can cause bacteremia, endocarditis, and urinary infection [51]. Although S. bovis is the 2nd greatest cause of infectious endocarditis from streptococci [50], it is frequently associated with gastrointestinal lesions, especially carcinoma of the colon [12,51-53]. Notably, the colonic neoplasia may arise years after the presentation of the condition of bacteremia or infectious endocarditis [12].

A retrospective review of forty-five documented cases of S bovis bacteremia was conducted by Gold et al. [12]. Subjects were identified by a search of computerized bacteriology records from one tertiary referral hospital and 1 community hospital located in the same city. Patient records were reviewed to identify the presence of colonic neoplasia, the use of gastrointestinal endoscopy, and the presence of gastrointestinal or extraintestinal malignancies. Seventeen patients (41% of adult patients) underwent colonoscopy. Colonic neoplasia was present in 16 patients (39% of adults). Invasive cancer was present in 13 patients (32% of adults), 8 of these had malignant lesions arising within the gastrointestinal tract, 3 affecting the colon and 5 patients had extraintestinal malignancies. The authors concluded that S. bovis bacteremia was associated with both colonic neoplasia and extracolonic malignancy.

It has been demonstrated that S. bovis or its wall extracted antigens (WEA) were able to promote carcinogenesis in rats [12]. In one of these investigations a total of 10 adult rats received i.p. injections of the carcinogen azoxymethane (AOM) (15 mg/kg body weight) once per week for 2 weeks. Fifteen days after the last injection of AOM (week 4) the rats were randomly divided into three groups. Twice per week during 5 weeks, the rats received, by gavage either S. bovis (1010 bacteria Group I), WEA (100 μg Group II) and controls (Group III).

One week after the last gavage (week 10), they found that administration of either S. bovis or its antigens promoted the progression of preneoplastic lesions. There were increased formations of hyperproliferative aberrant colonic crypts, enhanced expression of proliferation markers and increased production of IL-8 in the colonic mucosa. Normal rats treated with the bacteria did not develop hyperplastic colonic crypts, however. The authors concluded that S. bovis exerts its pathological activity in the colonic mucosa only when preneoplastic lesions are established.

Under identical experimental conditions Streptococcus gordonii was substituted for S. bovis. The number of preneoplastic lesions in the colon of S. gordonii-treated rats was similar to rats treated with AOM alone (22 ± 2). The authors suggested that S. bovis and its wall extracted antigens, unlike S. gordonii, act as promoters of carcinogenesis in a chemically-induced animal model.

In another investigation Biarc et al. [11] isolated 12 S. bovis cell-associated proteins (S300) and WEA. Cells of the human colonic epithelial cell line Caco-2 originally derived from an adenocarcinoma were grown to confluence and allowed to differentiate. These cells were stimulated with 200 ul of either S. bovis WEA (50 μg/ml) or cell-associated proteins S300 (100 μl).

The purified S300 fraction was able to trigger the human cell line and rat colonic mucosa to release chemokines (human IL-8 or rat CINC/GRO) and prostaglandin E2 (PgE2). The 12 S. bovis proteins were highly effective in the promotion of pre-neoplastic lesions in azoxymethane treated rats. In fact the S300 proteins were able to induce a 5-fold increase in PGE2 secretion from Caco-2 cells, as compared with cells stimulated with WEA. The study found that PGE2 release in the human cells correlated with an over-expression of cyclooxygease-2 (COX-2).

Evidence has shown that over-expression of COX-2 has a major role in mucosal inflammation [47] and is associated with inhibition of apoptosis [54] and enhancement of angiogenesis [55], which favor cancer initiation and development. It was reported by Biarc et al. [11] that S. bovis proteins also promoted cell proliferation by triggering mitogen-activated protein kinases (MAPKs), which can increase the incidence of cell transformation, the rate of genetic mutations and up-regulate COX-2. The investigators concluded that colonic bacteria such as S. bovis can contribute to cancer development particularly in chronic infection/inflammation diseases where bacterial components may interfere with cell function [11].

Genetic predisposition to cancer-causing infections

Research has shown that some populations are genetically predisposed to the infections that are associated with cancer and indeed have a higher risk of the cancer in question. The exact mechanisms remain unclear [38].

E. coli, crohn's disease and colon cancer

Inflammatory bowel disease (IBD) includes both ulcerative colitis (UC) and Crohn's disease (CD). Both of these disorders have an increased risk of colorectal cancer (CRC) [38,46,56]. Although colorectal cancer (CRC) in individuals with IBD only accounts for 1–2% of all cases of CRC in the general population, it is considered a serious complication of the disease and accounts for approximately 15% of all deaths in patients with IBD. The magnitude of the risk has been found to differ, however, in population-based studies [56-58]. Recent figures suggest that the risk of colon cancer for people with IBD increases by 0.5–1.0% yearly, 8–10 years after diagnosis. The magnitude of CRC risk also increases with early age at IBD diagnosis, longer duration of symptoms, and extent of disease, with pancolitis having more severe inflammation and a higher risk of dysplasia-carcinoma progression [56].

E. coli are found at higher levels in inflammatory bowel disease (IBD), therefore, studies have examined the mechanisms that may explain this phenomenon. A cell culture study by Martin et al [46] attempted to quantify and characterize mucosa-associated and intramucosal bacteria, particularly E. coli, in these inflammatory conditions. Their hypothesis was that the disease-associated alterations in mucosal glycosylation found in inflammatory bowel disease and colon cancer might predispose to altered recruitment of bacteria to the mucosa.

Mucosa-associated bacteria were isolated from biopsy samples of Crohn's disease, (n = 14); ulcerative colitis, (n = 21); noninflamed controls, (n = 24) and at surgical resection of colon cancer, (n = 21). Results found that mucosa-associated and intramucosal bacteria were cultured more commonly in Crohn's disease (79%, P = 0.03; and 71%, P < 0.01, respectively), and colon cancers (71% and 57%) than in noninflamed controls (42% and 29%) but not ulcerative colitis (38% and 48%). Mucosa-associated E. coli, which accounted for 53% of isolates, were more common in Crohn's disease (6/14; 43%) than in noninflamed controls (4/24, 17%), and intramucosal E. coli more common in Crohn's disease (29%; controls, 9%).

E. coli expressed hemagglutinins in 39% of Crohn's cases and 38% of cancers but only 4% of controls, and this correlated (P = 0.01) with adherence to embryonic intestinal cells (I407) and colon adenocarcinoma cells (HT29). Although close apposition of E. coli resulted in release of pro-inflammatory cytokines, cellular invasion by bacteria was not essential to this process [46].

Aspinell [59] suggested that the bacterial adherence found by Martin et al. [46] might result from activation of virulence genes following contact of the organisms with the inflamed mucosal cells. Martin et al. [46] found, however, that the mucosal isolates expressed of none of the known virulence genes, other than adherence genes. Martin and co-workers concluded that their findings supported a central role for mucosally adherent bacteria in the pathogenesis of Crohn's disease. They postulated that similar, lower grade, inflammatory changes could contribute to the risk of sporadic cancer development [46].

The authors stated however that it was certainly possible that the presence of the bacteria in the sub-mucus niche in human Crohn's disease and colon cancer could have been encouraged by disease-associated changes [46] in the mucosa. If true, their findings would result from colonization coincidental to the disease-associated alterations in mucosal glycosylation found in inflammatory bowel disease and colon cancer.

A study conducted by Masseret et al.[60] examined the E. coli strains isolated from patients with Crohn's disease (CD) with chronic ileal lesions (n = 14), early endoscopic recurrent lesions (n = 20), without endoscopic recurrence (n = 7), and controls (n = 21). Genetically linked E coli strains were isolated significantly more frequently from patients with chronic and recurrent CD (24/33 patients) than from controls (9/21) (p < 0.05). Most patients operated on for chronic ileal lesions (78.5%) harbored E coli strains belonging to the same cluster (p < 0.002 v controls). The prevalence of patients with early recurrent lesions harboring E coli strains belonging to this cluster was high but not significant. 21 of 26 strains isolated from patients with active CD demonstrated adherent ability to differentiated Caco-2 cells, indicating that most of the genetically related strains shared a common virulence trait. Comparison of E coli strains recovered from ulcerated and healthy mucosa of patients operated on for CD demonstrated in each patient that a single strain colonized the intestinal mucosa. The authors suggested that although a single E coli isolate was not found in Crohn's ileal mucosa, some genotypes were more likely than others to be associated with chronic or early recurrent ileal lesions.

S. typhi and susceptible populations

As previously stated, certain populations have an increased risk of gallbladder cancer (GC), however certain individuals may be predisposed to S. typhi infection which appears to increase the risk of GC. In an investigation by deJong et al. [61], three unrelated individuals with severe, idiopathic mycobacterial and Salmonella infections were found to lack IL-12Rβ1 chain expression. Interleukin-12 (IL-12) is a cytokine that promotes cell-mediated immunity to intracellular pathogens, such as S. typhi, by inducing type 1 helper T cell (TH1) responses and interferon-γ (IFN-γ) production. IL-12 binds to the high-affinity β1/β2 heterodimeric IL-12 receptor (IL-12R) complexes on T cell and natural killer cells. The cells of these patients were deficient in IL-12R signaling and IFN-γ production and their remaining T cell responses were independent of endogenous IL-12. IL-12Rβ1 sequence analysis revealed genetic mutations that resulted in premature stop codons in the extracellular domain. The genetic absence of IL12-Rβ1 expression represented an immune deficiency in these 3 patients. Interestingly, these patients did not develop any abnormal infections with other viral, bacterial, or fungal pathogens. The defect in IFN-production and extreme susceptibility to mycobacterial and Salmonella infections in these patients appeared to be a direct result of their lack of IL-12R expression and signaling. The authors concluded that selective susceptibility to mycobacterial and Salmonella infections, however, suggested that the type-1 cytokine pathway was essential for controlling resistance to the intracellular pathogens and that no redundant protective immune mechanism could compensate for this deficiency.

Respiratory conditions and increased susceptibility to lung cancer

75–90% of people who develop lung cancer are smokers, however, only a small proportion of smokers develop lung cancer [42]. Hence, epidemiological studies such as that of Littman et al [42] and Kocazeybek et al. [19] have been conducted to more closely identify risk factors. Identifying genetic factors that increase a smoker's risk of developing lung cancer may help scientists to better understand the etiology of lung cancer and more effectively target high-risk groups for screening. Additionally, genetic factors have been identified that appear to predict the prognosis of certain lung cancer patients [62]. For example, mutations affecting the epidermal growth factor receptor (EGFR) were significantly associated with specific genetic alterations. Supervised clustering analysis based on EGFR gene mutations elucidated a subgroup including all EGFR gene mutated tumors, which showed significantly shorter disease-free survival

To analyze the genetic alterations of primary lung adenocarcinoma in a high-throughput way, Shibata et al. [62] used laser-capture micro-dissection of cancer cells and array comparative genomic hybridization focusing on 800 chromosomal loci containing cancer-related genes. They identified a large number of chromosomal numerical alterations, including frequent amplifications. Three subgroups of lung adenocarcinoma were characterized by distinct genetic alterations and were associated with smoking history and gender. The authors concluded that multiple carcinogenic pathways exist; certain abnormalities appear related to gender and smoking while others may impact survival [62].

Bacterial strategies: cell cycle control and toxic warfare

Bacterial toxins can kill cells or at reduced levels alter cellular processes that control proliferation, apoptosis and differentiation. These alterations are associated with carcinogenesis and may either stimulate cellular aberrations or inhibit normal cell controls. Cell-cycle inhibitors, such as cytolethal distending toxins (CDTs) and the cycle inhibiting factor (Cif), block mitosis and are thought to compromise the immune system by inhibiting clonal expansion of lymphocytes. In contrast, cell-cycle stimulators such as the cytotoxic necrotizing factor (CNF) promote cellular proliferation and interfere with cell differentiation [20].

Bacterial toxins that subvert the host eukaryotic cell cycle have been classified as cyclomodulins. For example, CNF is a cell-cycle stimulator released by certain bacteria, such as E. coli. CNF triggers G1 – S transition and induces DNA replication. The number of cells does not increase, however. The cells become multinucleated instead, perhaps by the toxin's ability to inhibit cell differentiation and apoptosis [63,64].

Conversely the cytolethal distending toxin (CDT), as previously mentioned, is a cell-cycle inhibitor used by several species of Gram-negative bacteria, including Campylobacter jejuni and S. typhi. The CdtB unit of CDT is a DNAse that creates double-stranded DNA breaks causing cell cycle arrest, usually at the G2 checkpoint [65]. Cif is a cell cycle inhibitor found in enteropathogenic (EPEC) and enterohaemorrhagic (EHEC) E. coli. EPEC and EHEC deliver this novel toxin by injecting it into the infected epithelial cells. Cif arrests the cells at the G2/M phase [66]causing unique alterations in the host cell that result in attachment of the cytoskeleton to the host cell membrane. This anchoring of the cytoskeleton inhibits mitosis, causing cellular and nuclear enlargement. Although DNA synthesis is initiated it does not lead to nuclear division. Endoreduplicaton occurs resulting in cellular DNA content of 8–16n [20,66].

In a cell culture study, Haghjoo and Galán [65] found that S. typhi produced a unique cdtB-dependent CDT that required bacterial internalization into host cells. When Cos-2 cells were transfected with S. typhi the effects of the cdtB subunit were severe fragmentation of chromatin characteristic of the CdtB subunit of CDT expressed by other species. The authors proposed that S. typhi subsequent to internalization deviated from the usual endocytic pathway that leads to lysosomes, reaching an unusual membrane-bound compartment where it can survive and replicate. It is possible that this unique CDT may be involved in some aspects of the ability of S. typhi to cause long, persistent infections in humans, because, at least in other bacteria, this toxin has been shown to possess immunomodulatory activities.

Toxins are not the only strategy for evading the host's immune system, however. An early study by Kilian et al. [67] reported that some strains of Capnocytophaga ochracea, an oral pathogen, are capable of hydrolytically degrading immunoglobulin A subclass 1 found in the oral cavity. This property may enhance colonization and invasion of oral lesions which characterize many bacteremias due to Capnocytophaga species. [67]. Shurin et al. [68] obtained evidence that Capnocytophaga species inhibit polymorphonuclear leukocyte migration; a means by which these species may evade phagocytosis.

The immune system may also be evaded by the protection offered by bacterial biofilms. An example of this phenomenon is provided by uropathic E. coli species whose biofilms protect it from the immune system and making it difficult to treat these infections effectively by antibiotics. This has been demonstrated in bladder infections where the same species is recovered after repeated flare-ups thought to have been cleared by antibiotic therapy, suggesting a subclinical infection that has become chronic [69].

Bacterial site-specific colonization

Bacterial adherence is thought to be the first important step in colonization. It is now recognized that bacteria bind to and colonize host cells in a highly selective manner via a "lock- and key" mechanism. This selectivity of bacterial adhesion plays an important role in many infectious processes, and an understanding of the mechanisms involved could provide molecular explanations for the innate resistance or susceptibility of hosts and tissues to many infectious agents.

Regulators of complement activation (RCA proteins) prevent the destructive consequences of inappropriate immune activation. Decay-accelerating factor (CD55) is a member of the RCA protein family that protects host cells from complement damage and regulates the classical, alternative and lectin pathways that converge to target cells for destruction in all 3 pathways of the innate immune system [70]. CD55 is expressed on all serum-exposed cells. Perhaps due to its ubiquitous expression, it is thought that bacterial pathogens, including uropathogenic Escherichia coli, use CD55 as a receptor prior to infection. Williams et al. [70] suggested that pathogens have evolved to exploit the cellular roles of this molecule thereby gaining immunological advantage [70].

The influence on E. coli binding of the two known single amino acid polymorphisms within short consensus repeat (SCR) domains of CD55 was examined by Pham et al. [71] and Nowicki et al [72]. The bacterial strains sensitive to a change in SCR3 were found to be insensitive to changes in SCR4 and vice versa, suggesting that multiple, independent binding sites of CD55 were used by different bacterial strains. Evidence from those investigations suggested that E. coli strains sensitive to changes in one binding domain were not affected by changes in other domains. Furthermore, the use of CD55 as a receptor by a variety of uropathic E. coli was found to correlate with symptomatic infections [71,72]. Evidence from those investigations indicated the extraordinary degree of site-specific colonization of these closely related strains.

Bacteria associated with a coincidental or diagnostic role

Each year nearly 30,000 Americans are diagnosed with oral cancer [73,74]. Over 90% of these malignancies are oral squamous cell carcinoma (OSCC). Despite advances in surgery, radiation and chemotherapy, the five-year survival rate is 54%, one of the lowest of the major cancer sites and this rate has not improved significantly in recent decades [38,75,76]. The disease kills one person every hour – more people than cervical cancer, Hodgkin's disease, or malignant melanoma [38]. Notably, incidence in young adults (<40 years) is increasing in the U.S. [8,10] and worldwide [9,77]. The World Health Organization predicts a continuing worldwide increase in oral cancer over the next several decades [78].

Early detection followed by appropriate treatment, increases cure rates to about 80%, and greatly improves the quality of life by minimizing extensive, debilitating treatments [75]. Oral cancer is asymptomatic in its early stages, however, and in spite of the accessibility of the oral cavity to direct examination, these malignancies are often not detected until a late stage [79-81]. Oral cancer is unusual in that it carries a high risk of second primary tumors. Patients who survive a first cancer of the oral cavity have up to a 20-fold increased risk of developing a second primary oral cancer. The heightened risk can last 5–10 years, sometimes longer [82].

In response to the difficulties in effectively treating oral cancer, research studies are focusing on prevention and early diagnostics. Some of these studies have found that OSCC lesions are colonized by an altered microbiota [83,84]. Other investigations have found bacterial DNA or live organisms within oral cancer tissues [85,86]. The true nature of the relationships between oral bacteria and oral or esophageal cancers is, however, currently unknown.

PCR techniques have been used to seek the DNA of bacterial species in head and neck cancer tissues. Sasaki et al. [85] found S. anginosus DNA sequences in tissue samples from 127 cancer patients. Tissues examined included esophageal cancer, gastric cancer tissues, and dysplasia of the esophagus from esophageal cancer patients. No S. anginosus DNA was found in noncancerous esophagus or stomach samples. However, the degree of S. anginosus infection in biopsied tissues was much more obvious in the dysplastic and cancerous sections than in the noncancerous portions of the esophagus suggesting that S. anginosus infection occurred at an early stage of esophageal cancer. The authors suggested that S. anginosus could play a significant role in the carcinogenic process of most cases of esophageal cancer and some cases of gastric cancer by causing inflammation.

Morita et al. [86] found that 8 of 18 (44%) samples from the esophagus contained a detectable level of S. anginosus DNA, but only 5 of 38 (13%) of oral cancer had detectable DNA levels of this organism. The quantity of S. anginosus DNA in the esophageal cancer tissues was significantly higher than in oral cancer. The maximum amount of S. anginosus DNA was approximately 10 times higher in esophageal than in oral cancer tissues. In addition, none of the 5 different oral cancer sites (floor of mouth, maxillary or mandibular gingiva, buccal mucosa, and tongue) showed significant signs of S. anginosus infection. Most non-cancerous tissues of the esophagus and tongue showed an undetectable level of S. anginosus. The authors concluded that S. anginosus is associated with esophageal cancer, but is not closely related with oral cancer.

In a previous study by Mager et al. [87] it was determined that the salivary microbiota was similar to that of the oral soft tissues. Therefore, the investigators examined whether the salivary counts of 40 common oral bacteria in subjects with an oral squamous cell carcinoma (OSCC) lesion would differ from those found in cancer-free (OSCC-free) controls [83]. Unstimulated saliva samples were collected from 229 OSCC-free and 45 OSCC subjects and evaluated for their content of 40 common oral bacteria using checkerboard DNA-DNA hybridization.

DNA counts per ml saliva were determined for each species, averaged across subjects in the 2 subject groups, and the significance of differences between groups determined using the Mann-Whitney test and adjusted for multiple comparisons. The diagnostic sensitivity and specificity in detection of OSCC by levels of salivary organisms were computed and comparisons made separately between a non-matched group of 45 OSCC subjects and 229 controls and a group of 45 OSCC subjects and 45 controls matched by age, gender and smoking history.

Counts of 3 of the 40 species tested, Capnocytophaga gingivalis, Prevotella melaninogenica and Streptococcus mitis, were elevated in the saliva of individuals with OSCC (p < 0.001). When tested as diagnostic markers the 3 species were found to predict 80% of cancer cases (sensitivity) while excluding 83% of controls (specificity) in the non-matched group. Diagnostic sensitivity and specificity in the matched group were 80% and 82% respectively. These findings suggest that high salivary counts of C. gingivalis, P. melaninogenica and S. mitis could be diagnostic indicators of OSCC.

The reasons for the differences in colonization patterns of specific bacterial species at different host locations are only partially understood. These reasons include differences in nutrient availability, competition among species for binding sites, inter-species antagonisms or cooperations, and the differences in receptors present on different tissues that permit binding by specific adhesins possessed by different species. Other factors that may partly explain the unfavorable microbial shifts observed in oral carcinoma surface biofilms are a compromised host response or the irregularity of the lesion surface providing stagnant habitats.

The most intensely studied of these possibilities has been the specificity in adhesion of different bacterial species to receptors on oral soft tissues. Many studies have focused on fimbriae-mediated adhesion and adhesins in the adherence of different oral species to oral epithelial cells [88-91]. As a universal trait of cancer cells is alterations in cell surface receptors, studies have examined the colonization of healthy and cancerous epithelia [83,85-87,92].

A study by Neeser et al. [92] examined the binding of a common oral bacterial species, Streptococcus sanguis OMZ 9 to healthy and cancerous buccal cell lines. Results showed that S. sanguis bound to exfoliated human buccal epithelial cells in a sialic acid-sensitive manner. The desialylation of such cells invariably abolished adhesion of S. sanguis to the epithelial cell surface. The resialylation of desialylated HBEC with CMP-sialic acid and Galß1,3GalNAc α2,3-sialyltransferase specific for O-glycans restores the receptor function for S. sanguis OMZ 9, whereas a similar cell resialylation with the Galß1,4GlcNAc α2,6-sialyltmnsferase specific for N-glycans is without effect. These findings suggested that a 23 kDa cell surface glycoprotein bearing a carbohydrate sequence, NeuNAc alpha 2-3Gal beta 1-3GalNAc O-linked sugar chains, is recognized by S. sanguis on exfoliated human buccal epithelial cells. In similar experiments carried out with a buccal carcinoma cell line termed SqCC/Y1, S. sanguis did not attach in great numbers to cultured tumor cells. These cells were shown to not express the membrane glycoprotein bearing alpha 2,3-sialylated O-linked carbohydrate chains.

Aberrations in the cell surface carbohydrate structures have now been established as a universal characteristic of malignant transformation of cells, and cancer has been referred to as a molecular disease of the cell membrane glycoconjugates [93,94]. Thus, changes in the tumor cell surface structure could alter the adhesion of different species of oral bacteria. Notably, even species within the same genera, such as streptococci, have been found to differ in their colonization of healthy and cancerous oral tissues [83,87].

Bacteria and the prevention or treatment of cancer

Evidence is mounting that certain species of bacteria or their toxins may indeed have a protective or curative role in some cancers. Factors that would suggest a protective role of a bacterial species include: (1) colonization lowers the risk of a certain cancer; (2) elimination or absence of colonization raises the risk, or (3) introduction of the bacteria or its toxins cures or causes remission of the cancer.

Tumors and coley's toxins

Spontaneous tumor regression has followed severe bacterial, fungal, viral and protozoal infections. For hundreds of years this phenomenon inspired the development of the earliest cancer therapies. Reports of spontaneous remissions of advanced cancers infections can be found in the late nineteenth and early twentieth centuries. Many of these unexplained cures followed bacterial infections accompanied by high fevers.

An American surgeon, Dr. William Coley began the first well-documented use of bacteria and their toxins to treat end stage cancers. Coley first used live Streptococcus pyogenes cultures. Problems with the predictability of patient responses caused him to develop a safer vaccine in the late 1800's composed of two killed bacterial species, S. pyogenes and Serratia marcescens. In this way he could simulate an infection with the accompanying fever without the risk of an actual infection [95,96].

Coley's vaccine was widely used to successfully treat sarcomas, carcinomas, lymphomas, melanomas and myelomas. Complete, prolonged regression of advanced malignancy was documented in many cases. The combined reports of Coley and others estimated the 5-year survival rate at 80% in malignancies for which no treatment existed. Even in patients considered in the terminal stages of cancer some remarkable recoveries were reported with the patient often outliving the cancer [97].

Coley considered 4 points critical to success: (1) initiation of a naturally occurring infection with fever, (2) avoidance of immune tolerance by gradually increasing the dosage, (3) directly injecting the vaccine into the tumor when accessible, and (4) a minimum of 6 months of injections to avoid recurrences. Today little credence is given to the febrile response in fighting cancer [96,97].

A retrospective study was conducted in 1999 to compare the 10 year survival rate of patients treated by Coley's vaccine with modern conventional therapies. Richardson et al. [95] tried to match 128 of Coley's cases with 1,675 controls from the Surveillance Epidemiology End Result (SEER) cancer registry. The 2 populations were matched by age, gender, ethnicity, stage and radiation treatment status. Limitations included sample size and staging of patients receiving Coley's vaccine. The authors concluded that "Given the tremendous advances in surgical techniques and medicine in general, any cohort of modern patients should be expected to fare better than patients treated 50 or more years ago. Yet no such statistical advantage for the modern group was observed in this study." These findings were supported by case reports of spontaneous remissions or significant benefits when accidental infections occurred [98-100].

What role may a febrile response play in the remission of a tumor? Hobohm [101] offers the following hypothesis. Fever causes a cascade of events of inflammatory factors which activate resting dendritic cells (DC) that lead to the activation of T-cells. Cancer-cell specific T-cells usually remain in a state of anergy, most likely due to the absence of danger signals that usually accompany tissue destruction and inflammation upon acute infection [102]. A feverish bacterial infection may have a 3-fold beneficial effect. First, many infectious agents release endotoxins, like LPS, induce inflammatory cytokines and stimulate DC. Second, both thymocyte proliferation and generation of allo-specific CTL are increased with temperature in vitro [103]. Third, the vasculature of a tumor is more fragile than that of normal tissues and therefore more prone to destruction by the immune response. An infection causing hemorrhagic necrosis could trigger febrile collapse of the tumor vasculature [104,105]. Interestingly, the affinity of certain streptococci for binding to fibrinogen and fibrin may account for the 'homing' of bacterial enzymes to tumors as these cells are abundant in such proteins [106].

The mechanism by which infection cures cancer has been investigated. It has been suggested by Zacharski and Sukhatme [96] that the tumor regression observed by Coley and others is due to the activation of plasminogen. For example when the streptococcal spreading factor known as streptokinase (SK) combines with host plasminogen, plasmin is released. Plasmin triggers protease cascades that degrade plasma and extracellular matrix proteins. These mechanisms of degradation are toxic to tumor cells, disrupt the tumor extracellular matrix, alter tumor growth and inhibit metastasis [96]. The notion that plasminogen activators like SK might result in the remissions reported by Coley is appealing as they appear to spare healthy cells while attacking tumors. Zacharski et al. [107] hypothesized that although the potent enzymes produced by plasminogen activation may have a direct effect on cancer cells it was more likely they disrupted the cell-extracellular matrix of the tumor.

Investigators report that the traditional best treatment options for some candidate tumor types, such as advanced soft tissue sarcomas, breast cancer and melanoma, have not improved patient outcome substantially since Coley's day [96,108,109]. Currently, biologic response modifier therapies have moved beyond the nonspecific immunotherapy of Coley's era and laid the foundation for today's approaches. Zarcharski and Sukhatme [96] suggest that the early success of Coley's toxins are leading to therapies that engage the host's immune system against an individual's tumor offering new hope for cancer patients.

Autologous tumor cell vaccine therapy is an example of this new approach to cancer treatment. These vaccines differ markedly from conventional cytotoxic drug therapy that affect both normal and tumor cells. Tumor vaccines stimulate an individual's cell-mediated immune response by targeting the patient's tumor antigens. While efficacy of standard chemotherapy relates to the dose of the drug, the efficacy of a tumor vaccine is more complex, involving host-vaccine interactions [110]. These include: (1) immunogenicity of the vaccine regarding tumor-associated antigens as opposed to self; (2) the host's immune response in terms of immune recognition and effector mechanisms; and (3) the development of host systemic cell-mediated immunity, including long-term immunologic memory, (3–5 years). Therefore, the potency of the vaccine is not determined by immunogenicity alone but by its ability to induce the host anti-tumor response [110].

Bacillus calmette-guérin and autologous tumor cell vaccine vs. colon cancer

Certain tumor antigens are, however, normally weak immunogens. Therefore the use of adjuvants and the intradermal route of injection have, in some cases, produced an optimum antigenic vaccine. These adjuvant vaccines have induced effective host recognition of tumor-associated antigens and improved patient survival. For example, preliminary evidence by Hoover et al [111] suggested that active specific immunotherapy (ASI) of colon cancer using autologous tumor cell vaccines had potential in improving recurrence-free interval and survival. ASI assumes there are distinct tumor antigens on an individual's cancer cells that are either absent or in lower concentration on normal cells. The vaccine attempted to stimulate host's immune defenses against tumor-associated antigens by enhancing the immunogenicity of the patient's own tumor cells with an immunomodulating adjuvant, such as Bacillus Calmette-Guérin (BCG).

In a study by Hoover et al. [111] 80 eligible subjects with colon (47) or rectal (33) cancer were enrolled into a prospectively randomized, controlled clinical trial of active specific immunotherapy (ASI). An autologous tumor cell-Bacillus Calmette- Guerin (BCG) vaccine was used to determine whether ASI could improve disease- free status and survival. Eligible subjects had colon or rectal cancers extending through the bowel wall or had positive lymph nodes providing adequate cells from the primary tumor. Wide surgical removal of all tumors was performed with histologically proven clear margins and removal of involved lymph nodes. Prior to randomization individuals were screened for metastatic disease. Colon cancer and rectal cancer subjects were in separate but parallel studies and randomized into groups treated by resection alone or resection plus ASI. 3–4 weeks following surgery, both controls and treatment subjects were skin tested for immune competence and sensitivity to tuberculin purified protein derivative (PPD). Vaccines were begun in the ASI treatment group 4–5 weeks following surgery to allow for adequate immune recovery from surgery and anesthesia. A total of 24 colon and 17 rectal subjects composed the treatment group. With a median follow-up of 93 months, there was a significant improvement in survival (two-sided P = .02; hazards ratio, 3.97) and disease-free survival (two-sided P = .039; hazards ratio, 2.67) in all eligible colon cancer patients who received ASI. With a median follow-up of 58 months, no benefits were seen in patients with rectal cancer who received ASI. The authors concluded that the study suggested that ASI may be beneficial to patients with colon cancer.

In 2005, Uyl-de Groot et al. [110] conducted a multicenter, randomized controlled phase III clinical trial with Stage II and III colon cancer patients using ASI. Autologous tumor cells were used with the immunomodulating adjuvant Bacillus Calmette-Guérin (BCG) in a vaccine (OncoVAX®). Patients were randomized to receive either OncoVAX® or no therapy after surgical resection of the primary tumor. The vaccine was processed within 48 h after surgery in order to have viable, metabolically active, autologous tumor cells.

Analysis of prognostic benefit with a 5.8 year median follow-up, showed that the beneficial effects of OncoVAX® were statistically significant at all endpoints including recurrence-free interval, overall survival, and recurrence-free survival in Stage II colon cancer patients. Surgery alone cures 65% of Stage II colon cancer patients. For the remaining patients, OncoVAX® in an adjuvant setting significantly prolongs recurrence-free interval and significantly improves 5-year overall survival and recurrence-free survival. Unfortunately, no statistically significant prognostic benefits were achieved in Stage III patients [110].

Immunization with bacillus calmette-guérin vs. lung cancer

Grant et al [39] hypothesized that optimal chemotherapy with or without radiation followed by active immunization could eliminate microscopic residual disease and prolong survival. Immunization with GD3, a ganglioside expressed on the surface of most small cell lung cancers (SCLC) had not evoked a strong immune response. Therefore BEC2, a large xenogenic protein which mimics GD3, was judged to be a good immunogenic candidate. This approach had proven successful in extending the lives of melanoma patients [112].

Chapman et al. [113] conducted a phase II trial comparing 5 dose levels of BEC2. The study population consisted of 15 patients with small cell lung cancer (SCLC). All subjects had completed standard therapy and had achieved a partial or complete response. Patients received a series of five intradermal immunizations consisting of 2.5 mg of BEC2 plus BCG over a 10-week period. Blood was collected for serological analysis, and outcome was monitored. All patients developed anti-BEC2 antibodies, despite having received chemotherapy with or without thoracic radiation. Anti-GD3 antibodies were detected in five patients, including those with the longest relapse-free survival. The median relapse-free survival for patients with extensive stage disease was 10.6 months. In patients with limited stage disease a median relapse-free survival had not been reached with a follow-up of >47 months and only one of the 7 patients in this group relapsed. The authors reported that immunization of SCLC patients using BEC2 plus BCG after standard therapy could induce anti-GD3 antibodies and was safe. The survival and relapse-free survival in this group of patients was substantially better than those observed in similar patients receiving standard therapy.

A Phase III trial was conducted to evaluate BEC2 plus BCG as adjuvant therapy for limited small-cell carcinoma after chemotherapy and irradiation [114]. A total of 515 subjects were randomly assigned. Unfortunately, in this trial there was no improvement in survival, progression-free survival, or quality of life in subjects that were vaccinated. A trend toward prolonged survival was observed in the one third of subjects who developed a humoral response (p = 0.085), however.

The effectiveness of vaccines for several cancers was examined in a series of investigations. Xiang et al. [115] tested vaccines for human melanoma using the mutant S. typhi strain SL7207 as a DNA carrier. Tolerance against self-antigens was broken by genetically fusing ubiquitin with MHC I derivatives. Another approach coupled tumor-specific antibodies to functional IL-2. This combination in addition to oral vaccination with plasmid-encoded tumor antigens significantly enhanced protection against carcinoma of the colon [116], carcinoma of the lung [117] and melanoma [118]. Unstable cancer cells provided a challenge, however. Interestingly, this was overcome by targeting stable, proliferating endothelial cells of the tumor vasculature. This novel approach effectively inhibited angiogenesis [119].

Helicobacter pylori and esophageal adenocarcinoma

In industrialized countries the incidence of H pylori has been steadily decreasing [120]. The incidence of esophageal cancer (EA), however, is increasing [121]. Surprisingly, there is evidence that these two trends may be related. Several studies have determined that virulent strains of H pylori are found less commonly among patients with Barrett's esophagus and EA when compared with controls [122-124]. This led to studies that found positive associations among the increased incidence of obesity, GORD, Barrett's esophagus and EA [125].

Recently, a nested case-control study was conducted by de Martel et al [126] to assess the association between H. pylori infection and the risk of development of EA. Of a total of 128,992 members of an integrated health care system who had participated in a multiphasic health checkup (MHC) during 1964–1969, 52 patients developed EA during follow-up. Three randomly chosen control subjects from the MHC cohort were matched to each cancer subject, on the basis of age, gender, race, date and site of the MHC. Data on cigarette smoking, alcohol consumption, body mass index (BMI), and education level were obtained. Serum samples collected at the MHC were tested for IgG antibodies to H. pylori and to the H. pylori CagA antigen associated with H. pylori virulence.

Subjects with H. pylori infections were less likely than uninfected subjects to develop EA odds ratio (OR, 0.37) 95% confidence interval (CI, 0.16–0.88). This significant association was restricted to cancer subjects and control subjects <50 years old (OR, 0.20) (95% CI, 0.06–0.68). Interestingly, in patients with H. pylori infections, the OR for EA in those who tested positive for IgG antibodies to the CagA protein was similar to that for those who tested negative for it. BMI ≥25 and cigarette smoking, however, were strong independent risk factors for EA. The authors found, however, that the absence of H. pylori infection, independent of cigarette smoking and BMI, was associated with an increase in the risk of development of EA [126].

An epidemiological study in Sweden sought to determine whether BMI was associated with esophageal malignancies compared to gastric adenocarcinoma and controls. In a nationwide, population-based case-control study by Lagergren et al [127], between 1995 through 1997, a total of 189 patients with adenocarcinoma of the esophagus and 262 patients with adenocarcinoma of the gastric cardia were enrolled. These patients were compared with 167 patients with incident esophageal squamous cell carcinoma and 820 healthy controls.

Odds ratios were determined from BMI and cancer case-control status and ratios estimated the relative risk for the two adenocarcinomas studied. Calculations used multivariate logistic regression with adjustment for potential confounding factors. The adjusted odds ratio was 7.6 (95% CI, 3.8 to 15.2) among persons in the highest BMI quartile compared with persons in the lowest. Obese persons (persons with a BMI>30 kg/m2) had an odds ratio of 16.2 (CI, 6.3 to 41.4) compared with the leanest persons (persons with a BMI<22 kg/m2). The odds ratio for patients with cardia adenocarcinoma was 2.3 (CI, 1.5 to 3.6) in those in the highest BMI quartile compared with those in the lowest BMI quartile and 4.3 (CI, 2.1 to 8.7) among obese persons. Although a strong dose-dependent relation existed between BMI and esophageal adenocarcinoma, esophageal squamous-cell carcinoma was not associated with BMI. A modest but significant increase in intragastric acidity was also observed following the cure of H pylori infection which the authors postulated could contribute to gastroesophageal reflux disease (GORD).

The incidence of EA has increased rapidly over the last 30 years. During this period, the prevalence of Helicobacter pylori has decreased. Trends of increasing esophageal adenocarcinoma can be linked causally to increasing GORD which can be linked to an increasingly obese population. There appeared to be no plausible biological mechanism of association between H pylori, obesity, and GORD until studies of ghrelin, however.

Ghrelin was the first circulating hormone demonstrated to stimulate food intake in man. This peptide is produced in the stomach and regulates appetite, food intake, and body composition. The effects of ghrelin were examined in H pylori positive asymptomatic subjects by several investigators [128-130]. In a randomized double-blind cross-over study, by Wren et al. [129], ghrelin was shown to acutely enhance appetite and increase food intake in 9 healthy human subjects. There was a clear-cut increase in calories consumed by every individual from a free-choice buffet (mean increase 28 +/- 3.9%, p < 0.001) during ghrelin versus saline infusions. Furthermore, visual analogue scores for appetite were greater during ghrelin compared to saline infusion. Ghrelin had no effect on gastric emptying, however. The authors concluded that endogenous ghrelin was a potentially important new regulator of the complex systems controlling food intake and body weight.

Evidence is accumulating that ghrelin may explain the relative rarity of H. pylori among patients with Barrett's esophagus and EA. Findings from these studies and others support the notion that H. pylori may have a "protective" effect against EA [122,124]. Studies have found that curing H pylori infection increased plasma ghrelin in healthy asymptomatic subjects which may lead to increased appetite, weight gain and contribute to the increasing obesity seen in Western populations where the prevalence of H pylori is low. This evidence supports the notion that decreasing incidence of H pylori infection may lead to increased levels of plasma ghrelin and that this hormone appears to be a factor in increasing obesity which elevates the risk of GORD which is positively associated with Barrett's esophagus and increased risk of esophageal adenocarcinoma. It appears that the absence of H. pylori infection may be one of several factors that leads to the increased incidence in EA effect observed in Western populations.

The implications for treatment of individuals with H. pylori infection were addressed by Nakajima and Hattori [131]. They systematically reviewed the literature and estimated the expected annual incidence of esophageal or gastric cancer with and without eradication of H. pylori infection in patients with chronic atrophic gastritis. The expected annual incidence of gastric cancer in patients with corpus atrophy with persistent infection was at least 5.8-fold higher than that for esophageal adenocarcinoma after the eradication of infection at all ages. Even for patients with accompanying reflux esophagitis or Barrett's esophagus, the incidence of gastric adenocarcinoma with persistent infection was higher than that of esophageal adenocarcinoma after eradication of infection. The authors concluded, therefore, that if eradication of infection lowers the incidence of gastric cancer, it should be recommended for patients with corpus atrophy at all ages irrespective of the presence of reflux esophagitis or Barrett's esophagus, especially in populations having a high prevalence of gastric cancer [131].

In summary, increased BMI has been linked with the elimination of H. pylori infection. As the sphincter mechanism at the esophagogastric junction is weakened by weight it is not surprising that obese individuals have a higher incidence of gastric reflux or GORD [127]. GORD may lead to the development of Barrett's esophagus, which increases the risk of EA by 40-fold [132,133]. The study by Lagergren [127] provides evidence that these associations may be related as increasing body mass was associated with a stepwise increase in the risk of EA. If eradication of H. pylori infection lowers the incidence of gastric cancer, however, it should be recommended for patients with corpus atrophy at all ages irrespective of the presence of reflux esophagitis or Barrett's esophagus, especially in populations having a high prevalence of gastric cancer [131].
Attenuated bacteria: Promising carriers of DNA vaccines

Attenuated bacteria will enhance stimulation of the innate immune system yet increase the safety of a vaccine, [134] therefore they may be ideal for the delivery of vaccines. Animal studies have shown that attenuated S. typhimurium strains can successfully deliver a variety of genetically engineered DNA vaccine plasmids for therapeutic vaccination of mice against model tumors [117,118,135].

The identification of bacterial "carriers" for DNA vaccines that target cancer cells by site-specific colonization may allow the selective delivery of vaccine plasmids into tumor cells [136]. Colonization of these species may be considered coincidental to favorable conditions provided by the tumor yet prove clinically useful. Ultimately, however, the safety and efficacy of recombinant therapeutic agents expressed by plasmids must be conducted in appropriate animal models.

Conclusion

Cancer is commonly defined as the uncontrolled growth of abnormal cells that have accumulated enough DNA damage to be freed from the normal restraints of the cell cycle. Several pathogenic bacteria, particularly those that can establish a persistent, infection, can promote or initiate abnormal cell growth by evading the immune system or suppressing apoptosis [54,137]. Intracellular pathogens survive by evading the ability of the host to identify them as foreign. Other species or their toxins can alter host cell cycles or stimulate the production of inflammatory substances linked to DNA damage [120].

The highly site-specific adherence of bacteria involves binding species-specific adhesions to the required cell surface receptors. The role of species that colonize tumors could be causal, coincidental or potentially protective. If adhesion to the tumor in question is highly sensitive and specific it may be ideal not only in diagnosing the presence of a malignancy but also in delivering the appropriate therapy.

The bacterial species associated with cancer etiology are diverse; however, the infections they cause share common characteristics [18]. The time between acquiring the infection and cancer development is most often years or even decades as seen in cancers associated with H. pylori, S. typhi and S. bovis infections. Chronic interactions between the infective agent and immune response and/or a susceptible host appear to contribute to carcinogenesis [8,18,38,138]. Preventing or treating the infection may prevent the cancer in question. Notably, the vast majority of individuals infected with a cancer-causing species will not develop cancer [18].

Evidence suggests that certain individuals are more susceptible to infections linked to cancer development and that the incidence of certain cancers varies among populations. For example, gallbladder cancer is 3 times higher in females as in males in all populations [26]. Lung cancer is highest in populations that smoke however, only a small proportion of smokers develop lung cancer [42]. Although colon cancer is the 3rd highest cancer in the United States, individuals with IBD have a far greater risk of colorectal cancer than individuals without IBD [56-58].

A screening test for oral cancer based on salivary counts of bacterial species is appealing. Currently saliva is meeting the demand for inexpensive, non-invasive, and easy-to-use diagnostic aids for oral and systemic diseases, and for assessing risk behaviors such as tobacco and alcohol use. Although the colonization of certain bacterial species may be coincidental to favorable conditions provided by OSCC, increased numbers of certain salivary species may be clinically useful if shown to be a signature of oral cancer and if sensitivity and specificity are improved.

Successful treatment for cancers was reported by Dr. Coley and others one hundred years ago. His approach of using killed bacterial vaccines was surprisingly effective in some patients even in the latest stages of cancer. Dr. Coley believed that the human immune system had the power to cure cancers if properly stimulated. Today, some investigators agree and have designed new treatments that stimulate the immune system to recognize and target the lesion. Recent reports suggest that attenuated bacterial vaccines can safely and effectively deliver plasmids encoding tumor self antigens. These studies have reported successful treatment of certain cancers and prevention of recurrences [39,110,111]. Cancer vaccines although promising, present significant challenges. These include identification of highly effective bacterial strains and their attenuations, addressing safety issues and the problem of overcoming the peripheral T cell tolerance against tumor self-antigens [139]. Further, the response to vaccines will likely vary among individuals.

It appears that colonization by certain bacteria may reduce the risk of cancer in some populations. The epidemiological trends of esophageal adenocarcinoma and Helicobacter pylori infection have stimulated research into whether these may be coincidental or due to an inverse association. Intriguing results suggest there is an association represented by a complex continuum that begins with curing infections of virulent strains of H. pylori. The absence of H. pylori appears to elevate ghrelin which stimulates increased appetite in some individuals. High ghrelin levels appear to be associated with increased incidence of obesity. Obesity is reported to be a contributing factor in GORD. Finally, GORD may lead to Barrett's esophagus which increases the risk of esophageal adenocarcinoma. If these relationships can be proven, then the colonization of this species and its seemingly negative association with EA may be more clearly understood.

In summary, recent research has uncovered a great deal of information regarding the bacterial mechanisms used to cause, colonize or cure cancer, however, many questions remain. For example, do the bacteria in question initiate, promote, or merely show affinity for the neoplasm? Conversely does cancer weaken the host which facilitates acquiring the infection? Can the highly site specific colonization of certain bacteria for a tumor be clinically useful in diagnosis or treatment? Could attenuated bacteria be used in vaccines to safely and effectively deliver therapeutic agents? The continued exploration of these questions will bring research ever closer to the prevention, early diagnosis and truly effective treatment of this scourge of mankind.